Bernardo Rudy, Ph.D., M.D.

Mechanisms of Inhibition within the Telencephalon: "Where the Wild Things Are"
Fishell, Gord; Rudy, Bernardo
2011 ;34:535-567, Annual review of neuroscience
In this review, we first provide a historical perspective of inhibitory signaling from the discovery of inhibition through to our present understanding of the diversity and mechanisms by which GABAergic interneuron populations function in different parts of the telencephalon. This is followed by a summary of the mechanisms of inhibition in the CNS. With this as a starting point, we provide an overview describing the variations in the subtypes and origins of inhibitory interneurons within the pallial and subpallial divisions of the telencephalon, with a focus on the hippocampus, somatosensory, paleo/piriform cortex, striatum, and various amygdala nuclei. Strikingly, we observe that marked variations exist in the origin and numerical balance between GABAergic interneurons and the principal cell populations in distinct regions of the telencephalon. Finally we speculate regarding the attractiveness and challenges of establishing a unifying nomenclature to describe inhibitory neuron diversity throughout the telencephalon
— id: 134442, year: 2011, vol: 34, page: 535, stat: Journal Article,

Rapid developmental maturation of neocortical FS cell intrinsic excitability
Goldberg, Ethan M; Jeong, Hyo-Young; Kruglikov, Ilya; Tremblay, Robin; Lazarenko, Roman M; Rudy, Bernardo
2011 Mar;21(3):666-682, Cerebral cortex
Fast-spiking (FS) cells are a prominent subtype of neocortical gamma-aminobutyric acidergic interneurons that mediate feed-forward inhibition and the temporal sculpting of information transfer in neural circuits, maintain excitation/inhibition balance, and contribute to network oscillations. FS cell dysfunction may be involved in the pathogenesis of disorders such as epilepsy, autism, and schizophrenia. Mature FS cells exhibit coordinated molecular and cellular specializations that facilitate rapid responsiveness, including brief spikes and sustained high-frequency discharge. We show that these features appear during the second and third postnatal weeks driven by upregulation of K(+) channel subunits of the Kv3 subfamily. The low membrane resistance and fast time constant characteristic of FS cells also appears during this time, driven by expression of a K(+) leak current mediated by K(ir)2 subfamily inward rectifier K(+) channels and TASK subfamily 2-pore K(+) channels. Blockade of this leak produces dramatic depolarization of FS cells suggesting the possibility for potent neuromodulation. Finally, the frequency of FS cell membrane potential oscillations increases during development and is markedly slower in TASK-1/3 knockout mice, suggesting that TASK channels regulate FS cell rhythmogenesis. Our findings imply that some of the effects of acidosis and/or anesthetics on brain function may be due to blockade of TASK channels in FS cells
— id: 134292, year: 2011, vol: 21, page: 666, stat: Journal Article,

Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons
Rudy, Bernardo; Fishell, Gordon; Lee, Soohyun; Hjerling-Leffler, Jens
2011 Jan 1;71(1):45-61, Developmental Neurobiology
An understanding of the diversity of cortical GABAergic interneurons is critical to understand the function of the cerebral cortex. Recent data suggest that neurons expressing three markers, the Ca2+-binding protein parvalbumin (PV), the neuropeptide somatostatin (SST), and the ionotropic serotonin receptor 5HT3a (5HT3aR) account for nearly 100% of neocortical interneurons. Interneurons expressing each of these markers have a different embryological origin. Each group includes several types of interneurons that differ in morphological and electrophysiological properties and likely have different functions in the cortical circuit. The PV group accounts for approximately 40% of GABAergic neurons and includes fast spiking basket cells and chandelier cells. The SST group, which represents approximately 30% of GABAergic neurons, includes the Martinotti cells and a set of neurons that specifically target layerIV. The 5HT3aR group, which also accounts for approximately 30% of the total interneuronal population, is heterogeneous and includes all of the neurons that express the neuropeptide VIP, as well as an equally numerous subgroup of neurons that do not express VIP and includes neurogliaform cells. The universal modulation of these neurons by serotonin and acetylcholine via ionotropic receptors suggests that they might be involved in shaping cortical circuits during specific brain states andbehavioral contexts. (c) 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 45-61, 2011
— id: 115434, year: 2011, vol: 71, page: 45, stat: Journal Article,

DPP6 Establishes the A-Type K+ Current Gradient Critical for the Regulation of Dendritic Excitability in CA1 Hippocampal Neurons
Sun W.; Maffie J.; Lin L.; Petralia R.; Rudy B.; Hoffman D.
2011 ;71(6):1102-1115, Neuron
Subthreshold-activating A-type K⁺ currents are essential for the proper functioning of the brain, where they act to delay excitation and regulate firing frequency. In CA1 hippocampal pyramidal neuron dendrites, the density of A-type K⁺ current increases with distance from the soma, playing an important role in synaptic integration and plasticity. The mechanism underlying this gradient has, however, remained elusive. Here, dendritic recordings from mice lacking the Kv4 transmembrane auxiliary subunit DPP6 revealed that this protein is critical for generating the A-current gradient. Loss of DPP6 led to a decrease in A-type current, specifically in distal dendrites. Decreased current density was accompanied by a depolarizing shift in the voltage dependence of channel activation. Together these changes resulted in hyperexcitable dendrites with enhanced dendritic AP back-propagation, calcium electrogenesis, and induction of synaptic long-term potentiation. Despite enhanced dendritic excitability, firing behavior evoked by somatic current injection was mainly unaffected in DPP6-KO recordings, indicating compartmentalized regulation of neuronal excitability. 2011 Elsevier Inc
— id: 138726, year: 2011, vol: 71, page: 1102, stat: Journal Article,

The largest group of superficial neocortical GABAergic interneurons expresses ionotropic serotonin receptors
Lee, Soohyun; Hjerling-Leffler, Jens; Zagha, Edward; Fishell, Gord; Rudy, Bernardo
2010 Dec 15;30(50):16796-16808, Journal of neuroscience
A highly diverse population of neocortical GABAergic inhibitory interneurons has been implicated in multiple functions in information processing within cortical circuits. The diversity of cortical interneurons is determined during development and primarily depends on their embryonic origins either from the medial (MGE) or the caudal (CGE) ganglionic eminences. Although MGE-derived parvalbumin (PV)- or somatostatin (SST)-expressing interneurons are well characterized, less is known about the other types of cortical GABAergic interneurons, especially those of CGE lineage, because of the lack of specific neuronal markers for these interneuron subtypes. Using a bacterial artificial chromosome transgenic mouse line, we show that, in the somatosensory cortex of the mouse, the serotonin 5-hydroxytryptamine 3A (5-HT(3A)) receptor, the only ionotropic serotonergic receptor, is expressed in most, if not all, neocortical GABAergic interneurons that do not express PV or SST. Genetic fate mapping and neurochemical profile demonstrate that 5-HT(3A)R-expressing neurons include the entire spectrum of CGE-derived interneurons. We report that, in addition to serotonergic responsiveness via 5-HT(3A)Rs, acetylcholine also depolarizes 5-HT(3A)R-expressing neurons via nicotinic receptors. 5-HT(3A)R-expressing neurons in thalamocortical (TC) recipient areas receive weak but direct monosynaptic inputs from the thalamus. TC input depolarizes a subset of TC-recipient 5-HT(3A)R neurons as strongly as fast-spiking cells, in part because of their high input resistance. Hence, fast modulation of serotonergic and cholinergic transmission may influence cortical activity through an enhancement of GABAergic synaptic transmission from 5-HT(3A)R-expressing neurons during sensory process depending on different behavioral states
— id: 115436, year: 2010, vol: 30, page: 16796, stat: Journal Article,

Three groups of interneurons account for nearly 100% of neocortical GABAergic neurons
Rudy B; Fishell G; Lee S; Hjerling-Leffler J
2010 Nov 30;:?-?, Developmental Neurobiology
An understanding of the diversity of cortical GABAergic interneurons is critical to an understanding of the function of the cerebral cortex. Recent data suggest that neurons expressing three markers, the Ca2+ binding protein parvalbumin (PV), the neuropeptide somatostatin (SST) and the ionotropic serotonin receptor 5HT3a (5HT3aR) account for nearly 100% of neocortical interneurons. Interneurons expressing each of these markers have a different embryological origin. Each group includes several types of interneurons that differ in morphological and electrophysiological properties and likely have different functions in the cortical circuit. The PV group accounts for approximately 40% of GABAergic neurons and includes fast spiking basket cells and chandelier cells. The SST group, which represents approximately 30% of GABAergic neurons, includes the Martinotti cells and a set of neurons that specifically target layer IV. The 5HT3aR group, which also accounts for approximately 30% of the total interneuronal population, is heterogeneous and includes all of the neurons that express the neuropeptide VIP, as well as an equally numerous subgroup of neurons that don't express VIP and includes neurogliaform cells. The universal modulation of these neurons by serotonin and acetylcholine via ionotropic receptors suggests they might be involved in shaping cortical circuits during specific brain states and behavioral contexts. (c) 2010 Wiley Periodicals, Inc. Develop Neurobiol, 2010
— id: 149517, year: 2010, vol: , page: ?, stat: Journal Article,

Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes
Zagha, Edward; Manita, Satoshi; Ross, William N; Rudy, Bernardo
2010 Jun;103(6):3516-3525, Journal of neurophysiology
Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease
— id: 110076, year: 2010, vol: 103, page: 3516, stat: Journal Article,

Electrogenic tuning of the axon initial segment
Clark, Brian D; Goldberg, Ethan M; Rudy, Bernardo
2009 Dec;15(6):651-668, Neuroscientist
Action potentials (APs) provide the primary means of rapid information transfer in the nervous system. Where exactly these signals are initiated in neurons has been a basic question in neurobiology and the subject of extensive study. Converging lines of evidence indicate that APs are initiated in a discrete and highly specialized portion of the axon-the axon initial segment (AIS). The authors review key aspects of the organization and function of the AIS and focus on recent work that has provided important insights into its electrical signaling properties. In addition to its main role in AP initiation, the new findings suggest that the AIS is also a site of complex AP modulation by specific types of ion channels localized to this axonal domain
— id: 105978, year: 2009, vol: 15, page: 651, stat: Journal Article,

Quantitative analysis of neurons with Kv3 potassium channel subunits, Kv3.1b and Kv3.2, in macaque primary visual cortex
Constantinople, Christine M; Disney, Anita A; Maffie, Jonathan; Rudy, Bernardo; Hawken, Michael J
2009 ;516(4):291-311, Journal of comparative neurology
Voltage-gated potassium channels that are composed of Kv3 subunits exhibit distinct electrophysiological properties: activation at more depolarized potentials than other voltage-gated K+ channels and fast kinetics. These channels have been shown to contribute to the high-frequency firing of fast-spiking (FS) GABAergic interneurons in the rat and mouse brain. In the rodent neocortex there are distinct patterns of expression for the Kv3.1b and Kv3.2 channel subunits and of coexpression of these subunits with neurochemical markers, such as the calcium-binding proteins parvalbumin (PV) and calbindin D-28K (CB). The distribution of Kv3 channels and interrelationship with calcium-binding protein expression has not been investigated in primate cortex. We used immunoperoxidase and immunofluorescent labeling and stereological counting techniques to characterize the laminar and cell-type distributions of Kv3-immunoreactive (ir) neurons in macaque V1. We found that across the cortical layers almost-equal-to 25% of both Kv3.1b- and Kv3.2-ir neurons are non-GABAergic. In contrast, all Kv3-ir neurons in rodent cortex are GABAergic (Chow et al. [[1999]] J Neurosci. 19:9332-9345). The putatively excitatory Kv3-ir neurons were mostly located in layers 2, 3, and 4b. Further, the proportion of Kv3-ir neurons that express PV or CB also differs between macaque V1 and rodent cortex. These data indicate that, within the population of cortical neurons, a broader population of neurons, encompassing cells of a wider range of morphological classes may be capable of sustaining high-frequency firing in macaque V1.
— id: 105565, year: 2009, vol: 516, page: 291, stat: Journal Article,

The dipeptidyl-peptidase-like protein DPP6 determines the unitary conductance of neuronal Kv4.2 channels
Kaulin, Yuri A; De Santiago-Castillo, Jose A; Rocha, Carmen A; Nadal, Marcela S; Rudy, Bernardo; Covarrubias, Manuel
2009 Mar 11;29(10):3242-3251, Journal of neuroscience
The neuronal subthreshold-operating A-type K(+) current regulates electrical excitability, spike timing, and synaptic integration and plasticity. The Kv4 channels underlying this current have been implicated in epilepsy, regulation of dopamine release, and pain plasticity. However, the unitary conductance (gamma) of neuronal somatodendritic A-type K(+) channels composed of Kv4 pore-forming subunits is larger (approximately 7.5 pS) than that of Kv4 channels expressed singly in heterologous cells (approximately 4 pS). Here, we examined the putative novel contribution of the dipeptidyl-peptidase-like protein-6 DPP6-S to the gamma of native [cerebellar granule neuron (CGN)] and reconstituted Kv4.2 channels. Coexpression of Kv4.2 proteins with DPP6-S was sufficient to match the gamma of native CGN channels; and CGN Kv4 channels from dpp6 knock-out mice yielded a gamma indistinguishable from that of Kv4.2 channels expressed singly. Moreover, suggesting electrostatic interactions, charge neutralization mutations of two N-terminal acidic residues in DPP6-S eliminated the increase in gamma. Therefore, DPP6-S, as a membrane protein extrinsic to the pore domain, is necessary and sufficient to explain a fundamental difference between native and recombinant Kv4 channels. These observations may help to understand the molecular basis of neurological disorders correlated with recently identified human mutations in the dpp6 gene
— id: 94587, year: 2009, vol: 29, page: 3242, stat: Journal Article,

A novel DPP6 isoform (DPP6-E) can account for differences between neuronal and reconstituted A-type K(+) channels
Maffie, Jonathon; Blenkinsop, Timothy; Rudy, Bernardo
2009 Jan 16;449(3):189-194, Neuroscience letters
The channels mediating most of the somatodendritic A-type K(+) current in neurons are thought to be ternary complexes of Kv4 pore-forming subunits and two types of auxiliary subunits, the K(+) channel interacting proteins (KChIPs) and dipeptidyl-peptidase-like (DPPL) proteins. The channels expressed in heterologous expression systems by mixtures of Kv4.2, KChIP1 and DPP6-S resemble in many properties the A-type current in hippocampal CA1 pyramidal neurons and cerebellar granule cells, neurons with prominent A-type K(+) currents. However, the native currents have faster kinetics. Moreover, the A-type currents in neurons in intermediary layers of the superior colliculus have even faster inactivating rates. We have characterized a new DPP6 spliced isoform, DPP6-E, that produces in heterologous cells ternary Kv4 channels with very fast kinetics. DPP6-E is selectively expressed in a few neuronal populations in brain including cerebellar granule neurons, hippocampal pyramidal cells and neurons in intermediary layers of the superior colliculus. The effects of DPP6-E explain past discrepancies between reconstituted and native Kv4 channels in some neurons, and contributes to the diversity of A-type K(+) currents in neurons
— id: 94588, year: 2009, vol: 449, page: 189, stat: Journal Article,

Ternary Kv4.2 channels recapitulate voltage-dependent inactivation kinetics of A-type K+ channels in cerebellar granule neurons
Amarillo, Yimy; De Santiago-Castillo, Jose A; Dougherty, Kevin; Maffie, Jonathon; Kwon, Elaine; Covarrubias, Manuel; Rudy, Bernardo
2008 Apr 15;586(8):2093-2106, Journal of physiology
Kv4 channels mediate most of the somatodendritic subthreshold operating A-type current (I(SA)) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore-forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl-peptidase-like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX-S, or Kv4.2, KChIP1 and DPPX-S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the I(SA) in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX-S proteins in heterologous cells. The fact that this complex and unique behaviour (among A-type K(+) currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed-state inactivation and a weakly voltage-dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed-state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native I(SA)-mediating channels
— id: 79092, year: 2008, vol: 586, page: 2093, stat: Journal Article,

Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex
Ascoli, Giorgio A; Alonso-Nanclares, Lidia; Anderson, Stewart A; Barrionuevo, German; Benavides-Piccione, Ruth; Burkhalter, Andreas; Buzsaki, Gyorgy; Cauli, Bruno; Defelipe, Javier; Fairen, Alfonso; Feldmeyer, Dirk; Fishell, Gord; Fregnac, Yves; Freund, Tamas F; Gardner, Daniel; Gardner, Esther P; Goldberg, Jesse H; Helmstaedter, Moritz; Hestrin, Shaul; Karube, Fuyuki; Kisvarday, Zoltan F; Lambolez, Bertrand; Lewis, David A; Marin, Oscar; Markram, Henry; Munoz, Alberto; Packer, Adam; Petersen, Carl C H; Rockland, Kathleen S; Rossier, Jean; Rudy, Bernardo; Somogyi, Peter; Staiger, Jochen F; Tamas, Gabor; Thomson, Alex M; Toledo-Rodriguez, Maria; Wang, Yun; West, David C; Yuste, Rafael
2008 Jul;9(7):557-568, Nature reviews. Neuroscience
Neuroscience produces a vast amount of data from an enormous diversity of neurons. A neuronal classification system is essential to organize such data and the knowledge that is derived from them. Classification depends on the unequivocal identification of the features that distinguish one type of neuron from another. The problems inherent in this are particularly acute when studying cortical interneurons. To tackle this, we convened a representative group of researchers to agree on a set of terms to describe the anatomical, physiological and molecular features of GABAergic interneurons of the cerebral cortex. The resulting terminology might provide a stepping stone towards a future classification of these complex and heterogeneous cells. Consistent adoption will be important for the success of such an initiative, and we also encourage the active involvement of the broader scientific community in the dynamic evolution of this project
— id: 94591, year: 2008, vol: 9, page: 557, stat: Journal Article,

DPP6 Localization in Brain Supports Function as a Kv4 Channel Associated Protein
Clark, Brian D; Kwon, Elaine; Maffie, Jon; Jeong, Hyo-Young; Nadal, Marcela; Strop, Pavel; Rudy, Bernardo
2008 ;1:8-8, Frontiers in molecular neuroscience
The gene encoding the dipeptidyl peptidase-like protein DPP6 (also known as DPPX) has been associated with human neural disease. However, until recently no function had been found for this protein. It has been proposed that DPP6 is an auxiliary subunit of neuronal Kv4 K(+) channels, the ion channels responsible for the somato-dendritic A-type K(+) current, an ionic current with crucial roles in the regulation of firing frequency, dendritic integration and synaptic plasticity. This view has been supported mainly by studies showing that DPP6 is necessary to generate channels with biophysical properties resembling the native channels in some neurons. However, independent evidence that DPP6 is a component of neuronal Kv4 channels in the brain, and whether this protein has other functions in the CNS is still lacking. We generated antibodies to DPP6 proteins to compare their distribution in brain with that of the Kv4 pore-forming subunits. DPP6 proteins were prominently expressed in neuronal populations expressing Kv4.2 proteins and both types of protein were enriched in the dendrites of these cells, strongly supporting the hypothesis that DPP6 is an associated protein of Kv4 channels in brain neurons. The observed similarity in the cellular and subcellular patterns of expression of both proteins suggests that this is the main function of DPP6 in brain. However, we also found that DPP6 antibodies intensely labeled the hippocampal mossy fiber axons, which lack Kv4 proteins, suggesting that DPP6 proteins may have additional, Kv4-unrelated functions
— id: 94589, year: 2008, vol: 1, page: 8, stat: Journal Article,

K+ channels at the axon initial segment dampen near-threshold excitability of neocortical fast-spiking GABAergic interneurons
Goldberg, Ethan M; Clark, Brian D; Zagha, Edward; Nahmani, Mark; Erisir, Alev; Rudy, Bernardo
2008 May 8;58(3):387-400, Neuron
Fast-spiking cells (FS cells) are a prominent subtype of neocortical GABAergic interneurons with important functional roles. Multiple FS cell properties are coordinated for rapid response. Here, we describe an FS cell feature that serves to gate the powerful inhibition produced by FS cell activity. We show that FS cells in layer 2/3 barrel cortex possess a dampening mechanism mediated by Kv1.1-containing potassium channels localized to the axon initial segment. These channels powerfully regulate action potential threshold and allow FS cells to respond preferentially to large inputs that are fast enough to 'outrun' Kv1 activation. In addition, Kv1.1 channel blockade converts the delay-type discharge pattern of FS cells to one of continuous fast spiking without influencing the high-frequency firing that defines FS cells. Thus, Kv1 channels provide a key counterbalance to the established rapid-response characteristics of FS cells, regulating excitability through a unique combination of electrophysiological properties and discrete subcellular localization
— id: 79306, year: 2008, vol: 58, page: 387, stat: Journal Article,

Kv4 accessory protein DPPX (DPP6) is a critical regulator of membrane excitability in hippocampal CA1 pyramidal neurons
Kim, Jinhyun; Nadal, Marcela S; Clemens, Ann M; Baron, Matthew; Jung, Sung-Cherl; Misumi, Yoshio; Rudy, Bernardo; Hoffman, Dax A
2008 Oct;100(4):1835-1847, Journal of neurophysiology
A-type K+ currents have unique kinetic and voltage-dependent properties that allow them to finely tune synaptic integration, action potential (AP) shape and firing patterns. In hippocampal CA1 pyramidal neurons, Kv4 channels make up the majority of the somatodendritic A-type current. Studies in heterologous expression systems have shown that Kv4 channels interact with transmembrane dipeptidyl-peptidase-like proteins (DPPLs) to regulate the surface trafficking and biophysical properties of Kv4 channels. To investigate the influence of DPPLs in a native system, we conducted voltage-clamp experiments in patches from CA1 pyramidal neurons expressing short-interfering RNA (siRNA) targeting the DPPL variant known to be expressed in hippocampal pyramidal neurons, DPPX (siDPPX). In accordance with heterologous studies, we found that DPPX downregulation in neurons resulted in depolarizing shifts of the steady-state inactivation and activation curves, a shallower conductance-voltage slope, slowed inactivation, and a delayed recovery from inactivation for A-type currents. We carried out current-clamp experiments to determine the physiological effect of the A-type current modifications by DPPX. Neurons expressing siDPPX exhibited a surprisingly large reduction in subthreshold excitability as measured by a decrease in input resistance, delayed time to AP onset, and an increased AP threshold. Suprathreshold DPPX downregulation resulted in slower AP rise and weaker repolarization. Computer simulations supported our experimental results and demonstrated how DPPX remodeling of A-channel properties can result in opposing sub- and suprathreshold effects on excitability. The Kv4 auxiliary subunit DPPX thus acts to increase neuronal responsiveness and enhance signal precision by advancing AP initiation and accelerating both the rise and repolarization of APs
— id: 94590, year: 2008, vol: 100, page: 1835, stat: Journal Article,

Modulators of inhibitory synaptic transmission in mouse somatosensory cortex
Kruglikov, I; Rudy, B
2008 MAR ;18(5):S17-S17, European neuropsychopharmacology
— id: 78178, year: 2008, vol: 18, page: S17, stat: Journal Article,

Modulators of inhibitory synaptic transmission in mouse somatosensory cortex
Kruglikov, I; Rudy, B
2008 AUG ;18(6):S226-S227, European neuropsychopharmacology
— id: 90949, year: 2008, vol: 18, page: S226, stat: Journal Article,

Perisomatic GABA release and thalamocortical integration onto neocortical excitatory cells are regulated by neuromodulators
Kruglikov, Illya; Rudy, Bernardo
2008 Jun 26;58(6):911-924, Neuron
Neuromodulators such as acetylcholine, serotonin, and noradrenaline are powerful regulators of neocortical activity. Although it is well established that cortical inhibition is the target of these modulations, little is known about their effects on GABA release from specific interneuron types. This knowledge is necessary to gain a mechanistic understanding of the actions of neuromodulators because different interneuron classes control specific aspects of excitatory cell function. Here, we report that GABA release from fast-spiking (FS) cells, the most prevalent interneuron subtype in neocortex, is robustly inhibited following activation of muscarinic, serotonin, adenosine, and GABA(B) receptors--an effect that regulates FS cell control of excitatory neuron firing. The potent muscarinic inhibition of GABA release from FS cells suppresses thalamocortical feedforward inhibition. This is supplemented by the muscarinic-mediated depolarization of thalamo-recipient excitatory neurons and the nicotinic enhancement of thalamic input onto these neurons to promote thalamocortical excitation
— id: 80297, year: 2008, vol: 58, page: 911, stat: Journal Article,

Weighing the evidence for a ternary protein complex mediating A-type K+ currents in neurons
Maffie, Jonathon; Rudy, Bernardo
2008 Dec 1;586(Pt 23):5609-5623, Journal of physiology
The subthreshold-operating A-type K(+) current in neurons (I(SA)) has important roles in the regulation of neuronal excitability, the timing of action potential firing and synaptic integration and plasticity. The channels mediating this current (Kv4 channels) have been implicated in epilepsy, the control of dopamine release, and the regulation of pain plasticity. It has been proposed that Kv4 channels in neurons are ternary complexes of three types of protein: pore forming subunits of the Kv4 subfamily and two types of auxiliary subunits, the Ca(2+) binding proteins KChIPs and the dipeptidyl peptidase-like proteins (DPPLs) DPP6 (also known as DPPX) and DPP10 (4 molecules of each per channel for a total of 12 proteins in the complex). Here we consider the evidence supporting this hypothesis. Kv4 channels in many neurons are likely to be ternary complexes of these three types of protein. KChIPs and DPPLs are required to efficiently traffic Kv4 channels to the plasma membrane and regulate the functional properties of the channels. These proteins may also be important in determining the localization of the channels to specific neuronal compartments, their dynamics, and their response to neuromodulators. A surprisingly large number of additional proteins have been shown to modify Kv4 channels in heterologous expression systems, but their association with native Kv4 channels in neurons has not been properly validated. A critical consideration of the evidence suggests that it is unlikely that association of Kv4 channels with these additional proteins is widespread in the CNS. However, we cannot exclude that some of these proteins may associate with the channels transiently or in specific neurons or neuronal compartments, or that they may associate with the channels in other tissues
— id: 93219, year: 2008, vol: 586, page: 5609, stat: Journal Article,

Kv3.3 channels at the Purkinje cell soma are necessary for generation of the classical complex spike waveform
Zagha, Edward; Lang, Eric J; Rudy, Bernardo
2008 Feb 6;28(6):1291-1300, Journal of neuroscience
Voltage-gated potassium channel subunit Kv3.3 is prominently expressed in cerebellar Purkinje cells and is known to be important for cerebellar function, as human and mouse movement disorders result from mutations in Kv3.3. To understand these behavioral deficits, it is necessary to know the role of Kv3.3 channels on the physiological responses of Purkinje cells. We studied the function of Kv3.3 channels in regulating the synaptically evoked Purkinje cell complex spike, the massive postsynaptic response to the activation of climbing fiber afferents, believed to be fundamental to cerebellar physiology. Acute slice recordings revealed that Kv3.3 channels are required for generation of the repetitive spikelets of the complex spike. We found that spikelet expression is regulated by somatic, and not by dendritic, Kv3 activity, which is consistent with dual somatic-dendritic recordings that demonstrate spikelet generation at axosomatic membranes. Simulations of Purkinje cell Na+ currents show that the unique electrical properties of Kv3 and resurgent Na+ channels are coordinated to limit accumulation of Na+ channel inactivation and enable rapid, repetitive firing. We additionally show that Kv3.3 knock-out mice produce altered complex spikes in vitro and in vivo, which is likely a cellular substrate of the cerebellar phenotypes observed in these mice. This characterization presents new tools to study complex spike function, cerebellar signaling, and Kv3.3-dependent human and mouse phenotypes
— id: 76117, year: 2008, vol: 28, page: 1291, stat: Journal Article,

Distribution of Kv3.3 potassium channel subunits in distinct neuronal populations of mouse brain
Chang, Su Ying; Zagha, Edward; Kwon, Elaine S; Ozaita, Andres; Bobik, Marketta; Martone, Maryann E; Ellisman, Mark H; Heintz, Nathaniel; Rudy, Bernardo
2007 Jun 20;502(6):953-972, Journal of comparative neurology
Kv3.3 proteins are pore-forming subunits of voltage-dependent potassium channels, and mutations in the gene encoding for Kv3.3 have recently been linked to human disease, spinocerebellar ataxia 13, with cerebellar and extracerebellar symptoms. To understand better the functions of Kv3.3 subunits in brain, we developed highly specific antibodies to Kv3.3 and analyzed immunoreactivity throughout mouse brain. We found that Kv3.3 subunits are widely expressed, present in important forebrain structures but particularly prominent in brainstem and cerebellum. In forebrain and midbrain, Kv3.3 expression was often found colocalized with parvalbumin and other Kv3 subunits in inhibitory neurons. In brainstem, Kv3.3 was strongly expressed in auditory and other sensory nuclei. In cerebellar cortex, Kv3.3 expression was found in Purkinje and granule cells. Kv3.3 proteins were observed in axons, terminals, somas, and, unlike other Kv3 proteins, also in distal dendrites, although precise subcellular localization depended on cell type. For example, hippocampal dentate granule cells expressed Kv3.3 subunits specifically in their mossy fiber axons, whereas Purkinje cells of the cerebellar cortex strongly expressed Kv3.3 subunits in axons, somas, and proximal and distal, but not second- and third-order, dendrites. Expression in Purkinje cell dendrites was confirmed by immunoelectron microscopy. Kv3 channels have been demonstrated to rapidly repolarize action potentials and support high-frequency firing in various neuronal populations. In this study, we identified additional populations and subcellular compartments that are likely to sustain high-frequency firing because of the expression of Kv3.3 and other Kv3 subunits.
— id: 72705, year: 2007, vol: 502, page: 953, stat: Journal Article,

Differential regulation of action potential firing in adult murine thalamocortical neurons by Kv3.2, Kv1, and SK potassium and N-type calcium channels
Kasten, Michael R; Rudy, Bernardo; Anderson, Matthew P
2007 Oct 15;584(Pt 2):565-582, Journal of physiology
Sensory signals of widely differing dynamic range and intensity are transformed into a common firing rate code by thalamocortical neurons. While a great deal is known about the ionic currents, far less is known about the specific channel subtypes regulating thalamic firing rates. We hypothesized that different K(+) and Ca(2+) channel subtypes control different stimulus-response curve properties. To define the channels, we measured firing rate while pharmacologically or genetically modulating specific channel subtypes. Inhibiting Kv3.2 K(+) channels strongly suppressed maximum firing rate by impairing membrane potential repolarization, while playing no role in the firing response to threshold stimuli. By contrast, inhibiting Kv1 channels with alpha-dendrotoxin or maurotoxin strongly increased firing rates to threshold stimuli by reducing the membrane potential where action potentials fire (V(th)). Inhibiting SK Ca(2+)-activated K(+) channels with apamin robustly increased gain (slope of the stimulus-response curve) and maximum firing rate, with minimum effects on threshold responses. Inhibiting N-type Ca(2+) channels with omega-conotoxin GVIA or omega-conotoxin MVIIC partially mimicked apamin, while inhibiting L-type and P/Q-type Ca(2+) channels had small or no effects. EPSC-like current injections closely mimicked the results from tonic currents. Our results show that Kv3.2, Kv1, SK potassium and N-type calcium channels strongly regulate thalamic relay neuron sensory transmission and that each channel subtype controls a different stimulus-response curve property. Differential regulation of threshold, gain and maximum firing rate may help vary the stimulus-response properties across and within thalamic nuclei, normalize responses to diverse sensory inputs, and underlie sensory perception disorders
— id: 94592, year: 2007, vol: 584, page: 565, stat: Journal Article,

Differential characterization of three alternative spliced isoforms of DPPX
Nadal, Marcela S; Amarillo, Yimy; Vega-Saenz de Miera, Eleazar; Rudy, Bernardo
2006 Jun 13;1094(1):1-12, Brain research
Transient subthreshold-activating somato-dendritic A-type K(+) currents (I(SA)s) have fundamental roles in neuronal function. They cause delayed excitation, influence spike repolarization, modulate the frequency of repetitive firing, and have important roles in signal processing in dendrites. We previously reported that DPPX proteins are key components of the channels mediating these currents (Kv4 channels) (Nadal, M.S., Ozaita, A., Amarillo, Y., Vega-Saenz, E., Ma, Y., Mo, W., Goldberg, E.M., Misumi, Y., Ikehara, Y., Neubert, T.A., Rudy, B., 2003. The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron 37, 449-461). The DPPX gene encodes alternatively spliced transcripts that generate single-spanning transmembrane proteins with a short, divergent intracellular domain and a large extracellular domain. We characterized the modulatory effects on Kv4.2-mediated currents and the rat brain distribution of three splice variants of the DPPX subfamily of proteins. These three splice isoforms--DPPX-S, DPPX-L, and DPPX-K--are expressed in adult rat brain and modify the voltage dependence and kinetic properties of Kv4.2 channels expressed in Xenopus oocytes. Analysis of a deletion mutant that lacks the variable N-terminus showed that the N-terminus is not necessary for the modulation of Kv4 channels. Using in situ hybridization analysis, we found that the three splice variants are prominently expressed in brain regions where Kv4 subunits are also expressed. DPPX-K and DPPX-S mRNAs have a widespread distribution, whereas DPPX-L transcripts are concentrated in few specific areas of the rat brain. The emerging diversity of DPPX splice variants, differing only in the N-terminus of the protein, opens up intriguing possibilities for the modulation of Kv4 channels
— id: 68658, year: 2006, vol: 1094, page: 1, stat: Journal Article,

Developmental changes in the expression of calbindin and potassium-channel subunits Kv3.1b and Kv3.2 in mouse Renshaw cells
Song, ZM; Hu, J; Rudy, B; Redman, SJ
2006 APR ;139(2):531-538, Neuroscience
One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14. (C) 2006 published by Elsevier Ltd on behalf of IBRO
— id: 63856, year: 2006, vol: 139, page: 531, stat: Journal Article,

Developmental changes in the expression of calbindin and potassium-channel subunits Kv3.1b and Kv3.2 in mouse Renshaw cells (vol 139, pg 531, 2006)
Song, ZM; Hu, J; Rudy, B; Redman, SJ
2006 JUL ;141(1):543-543, Neuroscience
— id: 66448, year: 2006, vol: 141, page: 543, stat: Journal Article,

Specific functions of synaptically localized potassium channels in synaptic transmission at the neocortical GABAergic fast-spiking cell synapse
Goldberg, Ethan M; Watanabe, Shigeo; Chang, Su Ying; Joho, Rolf H; Huang, Z Josh; Leonard, Christopher S; Rudy, Bernardo
2005 May 25;25(21):5230-5235, Journal of neuroscience
Potassium (K+) channel subunits of the Kv3 subfamily (Kv3.1-Kv3.4) display a positively shifted voltage dependence of activation and fast activation/deactivation kinetics when compared with other voltage-gated K+ channels, features that confer on Kv3 channels the ability to accelerate the repolarization of the action potential (AP) efficiently and specifically. In the cortex, the Kv3.1 and Kv3.2 proteins are expressed prominently in a subset of GABAergic interneurons known as fast-spiking (FS) cells and in fact are a significant determinant of the fast-spiking discharge pattern. However, in addition to expression at FS cell somata, Kv3.1 and Kv3.2 proteins also are expressed prominently at FS cell terminals, suggesting roles for Kv3 channels in neurotransmitter release. We investigated the effect of 1.0 mM tetraethylammonium (TEA; which blocks Kv3 channels) on inhibitory synaptic currents recorded in layer II/III neocortical pyramidal cells. Spike-evoked GABA release by FS cells was enhanced nearly twofold by 1.0 mM TEA, with a decrease in the paired pulse ratio (PPR), effects not reproduced by blockade of the non-Kv3 subfamily K+ channels also blocked by low concentrations of TEA. Moreover, in Kv3.1/Kv3.2 double knock-out (DKO) mice, the large effects of TEA were absent, spike-evoked GABA release was larger, and the PPR was lower than in wild-type mice. Together, these results suggest specific roles for Kv3 channels at FS cell terminals that are distinct from those of Kv1 and large-conductance Ca2+-activated K+ channels (also present at the FS cell synapse). We propose that at FS cell terminals synaptically localized Kv3 channels keep APs brief, limiting Ca2+ influx and hence release probability, thereby influencing synaptic depression at a synapse designed for sustained high-frequency synaptic transmission
— id: 56149, year: 2005, vol: 25, page: 5230, stat: Journal Article,

International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels
Gutman, George A; Chandy, K George; Grissmer, Stephan; Lazdunski, Michel; McKinnon, David; Pardo, Luis A; Robertson, Gail A; Rudy, Bernardo; Sanguinetti, Michael C; Stuhmer, Walter; Wang, Xiaoliang
2005 Dec;57(4):473-508, Pharmacological reviews
— id: 72706, year: 2005, vol: 57, page: 473, stat: Journal Article,

Spontaneous Oscillatory Activity of Starburst Amacrine Cells in the Mouse Retina
Petit-Jacques, Jerome; Volgyi, Bela; Rudy, Bernardo; Bloomfield, Stewart
2005 Sep;94(3):1770-1780, Journal of neurophysiology
Using patch clamp techniques we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of ~3.5 Hz and an average maximal amplitude of ~120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by more than 70%, but reduced their frequency by about 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by CNQX, an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, subsequent application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells pre-stimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons
— id: 55982, year: 2005, vol: 94, page: 1770, stat: Journal Article,

ShK, a Pharmacological Tool for Studying Kv3.2 Channels
Yan, Lizhen; Herrington, James; Goldberg, Ethan; Dulski, Paula M; Bugianesi, Randal M; Slaughter, Robert S; Banerjee, Priya; Brochu, Richard M; Priest, Birgit T; Kaczorowski, Gregory J; Rudy, Bernardo; Garcia, Maria L
2005 May;67(5):1513-1521, Molecular pharmacology
Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. CHO-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. ShK, a peptide isolated from Stichodactyla helianthus venom, and a known high affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit (86)Rb(+) efflux from CHO-K1.hKv3.2b (IC50 of ~ 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus oocytes or in planar patch clamp studies, ShK inhibited hKv3.2b channels with IC50s of ~ 0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic beta cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with blockade of Kv3 channels, i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential afterhyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections. Taken together these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations
— id: 48219, year: 2005, vol: 67, page: 1513, stat: Journal Article,

Dipeptidyl peptidase 10 modulates Kv4-mediated A-type potassium channels
Zagha, Edward; Ozaita, Andres; Chang, Su Ying; Nadal, Marcela S; Lin, Udele; Saganich, Michael J; McCormack, Tom; Akinsanya, Karen O; Qi, Shu Y; Rudy, Bernardo
2005 May;280(19):18853-18861, Journal of biological chemistry
A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase IV (DPPIV) known as DPP10 was recently identified and linked to asthma susceptibility; however the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels. We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to the activity of DPPX, and significantly different than DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude and modified the voltage-dependence and kinetic properties of the current such that it resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization DPP10 was found to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10 and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity
— id: 48221, year: 2005, vol: 280, page: 18853, stat: Journal Article,

Potassium channel subunit Kv3.2 and the water channel aquaporin-4 are selectively localized to cerebellar pinceau
Bobik, Marketta; Ellisman, Mark H; Rudy, Bernardo; Martone, Maryann E
2004 Nov 12;1026(2):168-178, Brain research
The pinceau is a cerebellar structure formed by descending GABA-ergic basket cell axonal terminals converging on the initial axonal segment of Purkinje cell. Although basket cells exert a powerful inhibitory influence on the output of the cerebellar cortex, the function and mode of action of the pinceau are not understood because the majority of basket cell axons fail to make identifiable synaptic contacts with the Purkinje cell axon. Several proteins were previously reported to cluster specifically in this area, including a number of voltage-activated potassium channel subunits. In this study, we used immunohistochemistry, electron microscopy, and electron tomography to examine the ultrastructural localization of a novel voltage-gated potassium channel subunit, Kv3.2, in the pinceau. We found strong, selective localization of Kv3.2 to basket cell axons. Additionally, because potassium buffering is often conducted through water channels, we studied the extent of a brain-specific water channel, aquaporin-4 (AQP4), using confocal and electron microscopy. As expected, we found AQP4 was heavily localized to astrocytic processes of the pinceau. The abundance of potassium channels and AQP4 in this area suggests rapid ionic dynamics in the pinceau, and the unusual, highly specialized morphology of this region implies that the structural features may combine with the molecular composition to regulate the microenvironment of the initial segment of the Purkinje cell axon
— id: 48124, year: 2004, vol: 1026, page: 168, stat: Journal Article,

Inactivation gating of Kv4 K+ channels interacting with the dipeptidyl-aminopeptidase-like protein (DPPX)
Rocha, CA; Nadal, M; Rudy, B; Covarrubias, M
2004 JAN ;86(1):536A-536A, Biophysical journal
— id: 42461, year: 2004, vol: 86, page: 536A, stat: Journal Article,

ERG K !+ channels modulate the excitability of cortical neurons
Amarillo, Y.; Vega-Saenz de Miera, E. C.; Rudy, B.
2003 ;2003:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Ether-a-go-go related gene (erg) K+ channels contribute to the membrane repolarization during cardiac action potentials. Pharmacological blockage of erg K+ channels prevents firing frequency adaptation and unveils a contribution of these type of channels to the M current in cell lines with neuronal pedigree. Although erg K+ channel mRNAs are expressed prominently and widespread in the central nervous system (CNS), the presence of functional erg K+ channels in CNS neurons has not been demonstrated. We investigated the effects of selectively blocking erg K+ channels on the firing properties of somatosensory cortical neurons using whole-cell current clamp in acute mouse brain slices. Application of the erg K+ channel blocker WAY-123.398 (5-10 mM) (WAY) to pyramidal neurons of layers 2/3 (which prominently express erg3 mRNA) significantly increases the number of spikes fired by the neuron upon current injection. It also reduces spike frequency adaptation and afterhyperpolarization. In contrast, WAY has no effect on layer 6 pyramidal neurons (which do not express any erg channel mRNA) under the same conditions. Similar results are obtained with the use of the erg K+ channel blockers E-4031 and ergtoxin. We conclude that erg K+ channels are functionally expressed in cortical neurons and actively modulate their excitability
— id: 92524, year: 2003, vol: 2003, page: ?, stat: Journal Article,

International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels
Gutman, George A; Chandy, K George; Adelman, John P; Aiyar, Jayashree; Bayliss, Douglas A; Clapham, David E; Covarriubias, Manuel; Desir, Gary V; Furuichi, Kiyoshi; Ganetzky, Barry; Garcia, Maria L; Grissmer, Stephan; Jan, Lily Y; Karschin, Andreas; Kim, Donghee; Kuperschmidt, Sabina; Kurachi, Yoshihisa; Lazdunski, Michel; Lesage, Florian; Lester, Henry A; McKinnon, David; Nichols, Colin G; O'Kelly, Ita; Robbins, Jonathan; Robertson, Gail A; Rudy, Bernardo; Sanguinetti, Michael; Seino, Susumu; Stuehmer, Walter; Tamkun, Michael M; Vandenberg, Carol A; Wei, Aguan; Wulff, Heike; Wymore, Randy S
2003 Dec;55(4):583-586, Pharmacological reviews
This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/
— id: 42327, year: 2003, vol: 55, page: 583, stat: Journal Article,

Modulation of Kv4 K!+ channels by accesory proteins
Nadal, M. S.; Ozaita, A.; Chang, S.; Rudy, B.
2003 ;2003:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Kv4 proteins are thought to be the pore-forming subunits of the channels mediating most of the subthreshold-operating somatodendritic A-type K+ current (ISA) in neurons. Two types of proteins (KChIPs and DPPX) have been found that associate with Kv4 subunits and modulate channel properties. Both KChIPs and DPPX increase current magnitude, probably by facilitating the trafficking of Kv4 channel complexes to the plasma membrane. In addition, both subunits accelerate the recovery from inactivation and have effects on the voltage dependence of Kv4.2 channels, yet DPPX produces a more pronounced negative shift in both the conductance-voltage relation and in the voltage dependence of steady-state inactivation. The two types of protein differ on their effects on inactivation, while KChIP slows down the inactivation time course of Kv4 channels, DPPX accelerates the rate of channel inactivation, thus reproducing the fast kinetics of ISA channels in many neurons. The association of Kv4 proteins and KChIPs and DPPX has been demonstrated using co-immunoprecipitation assays. Quantitative immunoprecipitations show that only a small fraction of the KChIP or DPPX protein in brain is associated with Kv4.2 proteins, suggesting that KChIP and DPPX proteins may have Kv4 unrelated functions.We are currently investigating the effects of KChIPs and DPPX on the modulation of Kv4 channels by phorbol ester-mediated activation of PKC
— id: 92522, year: 2003, vol: 2003, page: ?, stat: Journal Article,

The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels
Nadal, Marcela S; Ozaita, Andres; Amarillo, Yimy; Vega-Saenz de Miera, Eleazar; Ma, Yuliang; Mo, Wenjun; Goldberg, Ethan M; Misumi, Yoshio; Ikehara, Yukio; Neubert, Thomas A; Rudy, Bernardo
2003 Feb 6;37(3):449-461, Neuron
Subthreshold-activating somatodendritic A-type potassium channels have fundamental roles in neuronal signaling and plasticity which depend on their unique cellular localization, voltage dependence, and kinetic properties. Some of the components of A-type K(+) channels have been identified; however, these do not reproduce the properties of the native channels, indicating that key molecular factors have yet to be unveiled. We purified A-type K(+) channel complexes from rat brain membranes and found that DPPX, a protein of unknown function that is structurally related to the dipeptidyl aminopeptidase and cell adhesion protein CD26, is a novel component of A-type K(+) channels. DPPX associates with the channels' pore-forming subunits, facilitates their trafficking and membrane targeting, reconstitutes the properties of the native channels in heterologous expression systems, and is coexpressed with the pore-forming subunits in the somatodendritic compartment of CNS neurons
— id: 38424, year: 2003, vol: 37, page: 449, stat: Journal Article,

The dipeptidyl aminopeptidase - like protein ( DPPX ) selectively modulates closed - state inactivation in Kv4 channels
Rocha, C.; Nadal, M. S.; Rudy, B.; Covarrubias, M.
2003 ;2003:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Somatodendritic A-type K+ currents mediated by Kv4 channels (ISA) exhibit preferential closed-state inactivation, which may be modulated by specific agents. DPPX accelerates the development of inactivation in Kv4 channels and is thought to be a key molecular component that is necessary to reconstitute the physiological properties of ISA. Here, we investigated the effect of DPPX on closed-state inactivation of heterologously expressed Kv4 channels (Kv4.1 and Kv4.3 in Xenopus oocytes). While macroscopic inactivation at positive membrane potentials (e.g., +70 mV) was modestly accelerated by DPPX, direct measurements of the development of closed-state inactivation in the presence of DPPX at hyperpolarized membrane potentials revealed dramatically reduced time constants for Kv4.1 and Kv4.3 (Fig. 1): 1.4+-0.2 s (control Kv4.1, at V1/2= -69 mV) and 0.2+-0.01 s (DDPX+Kv4.1, at V1/2= -81 mV), N=3; 5.2+-0.5 s (control Kv4.3, at V1/2= -67 mV) and 0.4+-0.08 s (DDPX+Kv4.3, at V1/2= -76 mV), N=4. Earlier studies from our laboratory suggested that internal sites in Kv4 channels control closed-state inactivation. Thus, DPPX may modulate Kv4 closed-state inactivation at an internal site
— id: 92523, year: 2003, vol: 2003, page: ?, stat: Journal Article,

MOLECULAR AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF THE CHANNELS UNDERLYING THE RESTING PERMEABILITY OF THALAMIC RELAY NEURONS
Amarillo, Y.; Vega-Saenz de Miera, E.; Rudy, B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
In thalamic relay neurons (TRNs) a change in the resting potential (VM) underlies a transition between two modes of firing which is critical for changes in thalamo-cortical transmission associated with physiological and pathological states of brain activity. The change in VM is largely produced by the blockade of a K+ current (IKleak) by neurotransmitters from ascending projections. We seek to identify the channels underlying the subthreshold behavior of TRNs using whole-cell and perforated patch recordings in acute mouse slices combined with molecular analysis of channel products expressed by TRNs. A hyperpolarization-activated cationic current (Ih), a persistent Na+ current and at least two components of K+ current contribute to the resting properties of TRNs. Ih contributes significantly to the VM of TRNs (blockade of Ih produces a apprx10 mV hyperopolarization). At least two inward rectifiers (probably mediated by Kir2.2 and GIRK channels) contribute an inward rectifying K+ current. Blockade of this current by low Ba2+ concentrations depolarizes TRNs by ltoreq 5 mV. The main effect of muscarinic agonists on TRNs is to block a linear K+ 'leak' component, which depolarizes the cell by >10 mV. The effects of volatile anesthetics on K2P channels and on IKleak suggest that K2P channels underlie the thalamic IKleak. The data suggests that TASK-1 channels are not responsible for the thalamic IKleak as they are in other neurons and that different channels mediate the resting permeability of TRNs of distinct thalamic nuclei
— id: 92525, year: 2002, vol: 2002, page: ?, stat: Journal Article,

MOLECULAR COMPONENTS OF THE CHANNELS UNDERLYING THE SUBTHRESHOLD - ACTIVATING A - TYPE K+ CURRENT
Nadal, M. S.; Ozaita, A.; Vega Saenz de M, E.; Amarillo, Y.; Lau, D.; Rudy, B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
The somato-dendritic subthreshold-activating A-type K+ current (ISA) is a fast K+ current that activates transiently at membrane potentials that are below the threshold for Na+ spike generation, and plays important roles in neuronal excitability. Two key components of the channels underlying most of the ISA have been identified: Kv4 pore-forming subunits (mainly Kv4.2 and Kv4.3) and KChIP associated proteins. We have recently obtained evidence for the presence in rat brain mRNA of transcripts encoding a novel factor (termed KAF), which accelerates the kinetics of Kv4 channels, and could explain the fast kinetics of native ISA in many neurons.) Immunoaffinity purification was used to purify native Kv4 channel complexes solubilized from rat cerebellar membranes. Three specific and prominent bands are observed when the complex is dissociated and separated by SDS-PAGE. Immunoblotting and sequencing has shown that two of these bands correspond to KChIP and Kv4 polypeptides, respectively. A third polypeptide(s) of apprx115 kDa co-purifies specifically with the channel complex and shows an abundance comparable to that of Kv4 and KChIP polypeptides, suggesting it is an important component of native Kv4 channels. Purification and sequencing of this polypeptide will be carried out to explore whether it is responsible for KAF activity. In addition, we are using a functional cloning procedure, suppression cloning, to clone cDNAs encoding KAF
— id: 92526, year: 2002, vol: 2002, page: ?, stat: Journal Article,

The physiological relevance of frequenin as a regulatory subunit of Kv4 channels
Nakamura, TY; Sturn, E; Pountney, DJ; Ozaita, A; Rudy, B; Coetzee, WA
2002 JAN ;82(1):125-125, Biophysical journal
— id: 105046, year: 2002, vol: 82, page: 125, stat: Journal Article,

Differential subcellular localization of the two alternatively spliced isoforms of the kv3.1 potassium channel subunit in brain
Ozaita, A; Martone, M E; Ellisman, M H; Rudy, B
2002 Jul;88(1):394-408, Journal of neurophysiology
Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons
— id: 32239, year: 2002, vol: 88, page: 394, stat: Journal Article,

Developmental expression of potassium-channel subunit Kv3.2 within subpopulations of mouse hippocampal inhibitory interneurons
Tansey, Emily Phillips; Chow, Alan; Rudy, Bernardo; McBain, Chris J
2002 ;12(2):137-148, Hippocampus
The developmental expression of the voltage-gated potassium channel subunit, Kv3.2, and its localization within specific mouse hippocampal inhibitory interneuron populations were determined using immunoblotting and immunohistochemical techniques. Using immunoblotting techniques, the Kv3.2 protein was weakly detected at postnatal age day 7 (P7), and full expression was attained at P21 in tissue extracts from homogenized hippocampal preparations. A similar developmental profile was observed using immunohistochemical techniques in hippocampal tissue sections. Kv3.2 protein expression was clustered on the somata and proximal dendrites of presumed inhibitory interneurons. Using double immunofluorescence, Kv3.2 subunit expression was detected on subpopulations of GABAergic inhibitory interneurons. Kv3.2 was detected in approximately 100% of parvalbumin-positive interneurons, 86% of interneurons expressing nitric oxide synthase, and approximately 50% of somatostatin-immunoreactive cells. Kv3.2 expression was absent from both calbindin- and calretinin-containing interneurons. Using immunoprecipitation, we further demonstrate that Kv3.2 and its related subunit Kv3.1b are coexpressed within the same protein complexes in the hippocampus. These data demonstrate that potassium channel subunit Kv3.2 expression is developmentally regulated in a specific set of interneurons. The vast majority of these interneuron subpopulations possess a 'fast-spiking' phenotype, consistent with a role for currents through Kv3.2 containing channels in determining action potential kinetics in these cells
— id: 48132, year: 2002, vol: 12, page: 137, stat: Journal Article,

MODIFICATION OF KV2.1 K!+ CURRENTS BY THE SILENT KV10 SUBUNITS
Vega, E. C.; Rudy, B.
2002 ;2002:?-?, Society for Neuroscience Abstract Viewer & Itinerary Planner
Channels containing Kv2 K+ channel subunits are thought to underlie most of the sustained K+ currents in CNS neurons. Several silent pore-forming subunits interact with Kv2 proteins to produce functional delayed rectifier K+ channels with modified electrophysiological or pharmacological properties. Here we present the cloning and characterization of two novel Kv2 interacting silent pore forming subunits, Kv10.1a and Kv10.1b, from human and rat. These alternatively-spliced variants arise by an alternative splice site in exon 1. The transcripts encode proteins with 436 and 425 amino acids (aa) in human and 433 and 422 aa in rat and mouse. The difference between the two spliced variants consist of a 9 aa insert between the 1st and 2nd transmembrane domains. Expression of Kv10 mRNAs in human was detected by Northern blot analysis in brain, kidney, lung, and pancreas. In human brain Kv10 mRNAs were expressed in cortex, hippocampus, caudate, putamen, amygdala and weakly in substantia nigra. but not in cerebellum, medulla, spinal cord, corpus callosum or thalamus. In rat, Kv10s were detected in brain and adrenal gland. In situ hybridization in rat brain sections demonstrates Kv10 mRNA expression in cortex, hippocampus, caudate-putamen and amygdala. No current was observed in Xenopus oocytes injected with Kv10 cRNAs alone. Co-injection of Kv2.1 and Kv10.1a or b decreased the amount of current expressed, as compared to oocytes injected with Kv2.1 cRNA alone, and reduces the inactivation rate without any appreciable change in other electrophysiological or pharmacological properties. The Kv10 gene maps to human chromosome 2p22.1
— id: 92527, year: 2002, vol: 2002, page: ?, stat: Journal Article,

Sleep EEG in mice that are deficient in the potassium channel subunit K.v.3.2
Vyazovskiy, Vladyslav V; Deboer, Tom; Rudy, Bernardo; Lau, David; Borbely, Alexander A; Tobler, Irene
2002 Aug 30;947(2):204-211, Brain research
Voltage-gated potassium channels containing the K.v.3.2 subunit are expressed in specific neuronal populations such as thalamocortical neurons and fast spiking GABAergic interneurons of the neocortex and hippocampus. These K(+)-channels play a major role in the regulation of firing properties in these neurons. We investigated whether the K.v.3.2 subunit contributes to the generation of the sleep electroencephalogram (EEG). The EEG of a frontal and occipital derivation of K.v.3.2-deficient mice and littermate controls was recorded during a 24-h baseline, 6-h sleep deprivation (SD) and subsequent 18-h recovery to assess also the effects of the K.v.3.2 subunit deficiency under physiological sleep pressure. The K.v.3.2-deficient mice had lower EEG power density in the frequencies between 3.25 and 6 Hz in nonREM (NREM) sleep and 3.25-5 Hz in REM sleep. These differences were more prominent in the frontal derivation than in the occipital derivation. The waking EEG spectrum was not affected by the deletion. In both genotypes SD induced a prominent increase in slow-wave activity in NREM sleep (mean EEG power density between 0.75 and 4.0 Hz), and a concomitant decrease in sleep fragmentation. The effects of SD did not differ significantly between the genotypes. The results indicate that K.v.3.2 channels may be involved in the generation of EEG oscillations in the high delta and low theta range in sleep. They support the notion that GABA-mediated synchronization of cortical activity contributes to the electroencephalogram
— id: 48131, year: 2002, vol: 947, page: 204, stat: Journal Article,

Synchronization of gamma band oscillations in mice
Harvey, M. A.; Lau, D.; Rudy, B.; de Yebenes, E. Garcia; Contreras, D.
2001 ;27(1):121-121, Abstracts (Society for Neuroscience)
Synchronized gamma band oscillations have been implicated in a number of neural processes including, attention, and sensory coding. The mechanism(s) for generation and synchronization of these oscillations are unknown. A central question is how distant ensembles of neurons are brought into synchrony despite conduction delays. One recent model demonstrates that such synchrony can be attained by evoking spike doublets (high frequency pairs of action potentials) in hippocampal interneurons, (Traub et al. Nature: 383, 621 1996). The duration of doublets are modulated by the phase relationships between the activity of distant and local excitatory populations, and serve as an error signal to bring these populations into synchrony. It has recently been demonstrated, (Lau et al. J. Neurosci: 15, 9071 2000), that such high frequency firing in cortical interneurons in part depends upon the presence of a particular voltage gated potassium channel (Kv3.2). This channel has properties that allow for a rapid repolarization of the membrane potential necessary for the generation of spike doublets. We show that Kv3.2 knockout mice are impaired in their ability to temporally organize gamma frequency oscillations in the cerebral cortex. Using arrays of electrodes spanning 2.5 mm of cortex we recorded field activity in anesthetized, and awake behaving mice. Spatiotemporal analysis performed across our array of electrodes revealed a significant reduction in the ability of Kv3.2-/- mice to synchronize distant neuronal populations at gamma frequencies. Such deficits were not evident when comparing local ensembles nor for slow oscillations
— id: 92531, year: 2001, vol: 27, page: 121, stat: Journal Article,

Modulation of Kv3 potassium channels expressed in CHO cells by a nitric oxide-activated phosphatase
Moreno H; Vega-Saenz de Miera E; Nadal MS; Amarillo Y; Rudy B
2001 Feb 1;530(Pt 3):345-358, Journal of physiology
1.Voltage-gated K+ channels containing Kv3 subunits play specific roles in the repolarization of action potentials. Kv3 channels are expressed in selective populations of CNS neurons and are thought to be important in facilitating sustained and/or repetitive high frequency firing. Regulation of the activity of Kv3 channels by neurotransmitters could have profound effects on the repetitive firing characteristics of those neurons. 2.Kv3 channels are found in several neuronal populations in the CNS that express nitric oxide synthases (NOSs). We therefore investigated whether Kv3 channels are modulated by the signalling gas nitric oxide (NO). 3.We found that Kv3.1 and Kv3.2 currents are potentially suppressed by D-NONOate and other NO donors. The effects of NO on these currents are mediated by the activation of guanylyl cyclase (GC), since they are prevented by Methylene Blue, an inhibitor of GC, and by ODQ, a specific inhibitor of the soluble form of GC. Moreover, application of 8-Br-cGMP, a permeant analogue of cGMP, also blocked Kv3.1 and Kv3.2 currents. 4.KT5283, a cGMP-dependent protein kinase (PKG) blocker, prevented the inhibition of Kv3.1 and Kv3.2 currents by D-NONOate and 8-Br-cGMP. This indicates that activation of PKG as a result of the increase in intracellular cGMP levels produced by D-NONOate or 8-Br-cGMP is necessary for channel block. 5.Although the effects of NO on Kv3.1 and Kv3.2 channels require PKG activity, two observations suggest that they are not mediated by phosphorylation of channel proteins: (a) the reagents affect both Kv3.2 and Kv3.1 channels, although only Kv3.2 proteins have a putative PKA-PKG phosphorylation site, and (b) mutation of the PKA-PKG phosphorylation site in Kv3.2 does not interfere with the effects of NO or cGMP. 6.The inhibitory effects of NO and cGMP on Kv3.1 and Kv3.2 currents appear to be mediated by the activation of serine-threonine phosphatase, since they are blocked by low doses of okadaic acid. Furthermore, direct intracellular application of the catalytic subunit of protein phosphatase 2A inhibited Kv3.2 currents, indicating that activity of PKG-induced phosphatase is necessary and sufficient to inhibit these channels. 7.The results suggest that basal phosphorylation of Kv3 channel proteins is required for proper channel function. Activation of phosphatases via NO or other signals that increase cGMP might be a potent mechanism to regulate Kv3 channel activity in neurons
— id: 18827, year: 2001, vol: 530, page: 345, stat: Journal Article,

Evidence for the presence of a novel Kv4-mediated A-type K(+) channel-modifying factor
Nadal MS; Amarillo Y; Vega-Saenz de Miera E; Rudy B
2001 Dec 15;537(Pt 3):801-809, Journal of physiology
1. Subthreshold-operating transient (A-type) K(+) currents (I(SA)s) are important in regulating neuronal firing frequency and in the modulation of incoming signals in dendrites. It is now known that Kv4 proteins are the principal, or pore-forming, subunits of the channels mediating I(SA)s(.) In addition, accessory subunits of Kv4 channels have also been identified. These either have no effect or slow down the inactivation kinetics of Kv4 channels. However, in many neuronal populations the I(SA) is faster, not slower, than the current generated by channels containing only Kv4 proteins. 2. Evidence is presented for the presence in rat cerebellar mRNA of transcripts encoding a molecular factor, termed KAF, that accelerates the kinetics of Kv4 channels. Size-fractionation of cerebellar mRNA in sucrose gradients separated the high molecular weight mRNAs (4-7 kb) encoding KAF from the low molecular weight ones (1.5-3 kb) encoding factors that slow down the inactivation kinetics of Kv4 channels. The latter were identified as KChIPs using anti-KChIP antisense oligonucleotides. 3. Both anti-KChIP and anti-Kv4 antisense oligonucleotides failed to eliminate KAF's activity from the high molecular weight mRNA fraction, thus suggesting that KAF might be a novel subunit(s) that can contribute to generating native I(SA) channel diversity. 4. The time course of the currents expressed by KAF-modified Kv4 channels resembles more closely the time course of the native I(SA) in cerebellar granule cells
— id: 39465, year: 2001, vol: 537, page: 801, stat: Journal Article,

Modulation of Kv4 potassium channels by a novel molecular component
Nadal, M. S.; Amarillo, Y.; Vega-Saenz de Miera, E. C.; Rudy, B.
2001 ;27(2):2145-2145, Abstracts (Society for Neuroscience)
Subthreshold-operating transient A-type currents (ISA) cause delayed excitation, may affect action potential repolarization, and influence the duration of the interspike interval. Antisense hybrid arrest of rat brain mRNA and other gene elimination methods have shown that subunits of the voltage-gated potassium subfamily Kv4 are the pore-forming subunits of native ISA channels. Expression of Kv4 proteins in Xenopus oocytes produces a current that resembles ISA, yet native ISAs produced by poly(A) RNA from rat brain have different kinetics. This discrepancy can be explained by the presence of auxiliary subunits. However, co-expression of members of a group of such auxiliary subunits, named KChIPs, and Kv4s does not render A-currents with the kinetics of the native currents. We size-fractionated poly(A)+ RNA from rat cerebellum and found a 4-7 kb RNA fraction that encodes a factor(s) that modifies the kinetics of A-currents expressed by Kv4.2 cRNA. Co-injection of Kv4.2 with this fraction accelerates the activation and inactivation of Kv4 channels, produces a 20 mV negative shift in the conductance voltage relation and accelerates recovery from inactivation. This activity cannot be eliminated by hybrid arrest with antisense degenerated oligonucleotides against Kv4 subunits, suggesting that the novel factor is not a new member of the Kv4 subfamily. Antisense treatment with oligonucleotides complementary to KChIP sequences also failed to arrest the activity. We propose that this factor is a novel component of native A-type currents, which contributes to generating subthreshold-operating A-channel functional diversity
— id: 92529, year: 2001, vol: 27, page: 2145, stat: Journal Article,

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2
Nakamura TY; Nandi S; Pountney DJ; Artman M; Rudy B; Coetzee WA
2001 Jun 22;499(3):205-209, FEBS letters
The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels
— id: 21167, year: 2001, vol: 499, page: 205, stat: Journal Article,

A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents
Nakamura TY; Pountney DJ; Ozaita A; Nandi S; Ueda S; Rudy B; Coetzee WA
2001 Oct 23;98(22):12808-12813, Proceedings of the National Academy of Sciences of the United States of America
Frequenin, a Ca(2+)-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K(+) currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca(2+)-dependent manner. It also accelerates recovery from inactivation. Closely related Ca(2+)-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca(2+)-sensitive regulatory component of native A-type K(+) currents
— id: 25507, year: 2001, vol: 98, page: 12808, stat: Journal Article,

Different effects of the Ca2+-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2
Nakamura, TY; Pountney, DJ; Nandi, S; Artman, M; Rudy, B; Coetzee, WA
2001 JUN ;33(6):A83-A83, Journal of molecular & cellular cardiology
— id: 54847, year: 2001, vol: 33, page: A83, stat: Journal Article,

Localization of Kv3 potassium channel subunits in the mouse retina
Ozaita, A.; Volgyi, B.; Bloomfield, S. A.; Rudy, B.
2001 ;27(2):2148-2148, Abstracts (Society for Neuroscience)
Kv3 K+ channels have been shown to play critical roles in fast spike repolarization and in enabling high frequency firing in cortical interneurons and in brainstem auditory neurons. We examined the cellular and subcellular distribution of Kv3.1a, Kv3.1b and Kv3.2 subunits in mouse retina. Expression of Kv3.1b was detected in few ganglion cells and two types of amacrine cells. One type had small somata and were distributed in mirror symmetry in the inner nuclear layer (INL) and the ganglion cell layer (GCL), in neurons projecting to strata 2 and 4 of the inner plexiform layer (IPL). These cells coexpressed Kv3.1b, calretinin and choline acetyltransferase. This group of Kv3.1b expressing neurons is both morphologically and neurochemically identical to the starburst amacrine cells. Interestingly, Kv3.1a immunoreactivity was restricted to the dendritic processes of the starburst cells in strata 2 and 4. Kv3.2 subunits were detected in the somata of a few ganglion cells, in scattered amacrine cell bodies and proximal dendrites located in the INL and in diffuse fiber-like structures throughout the IPL. Thus, Kv3.1b and Ky3.2 showed both somatic and dendritic localization in retinal amacrine and ganglion cells whereas Kv3.1a subunits were restricted to the processes in the IPL. These results indicate that Kv3.1 and Kv3.2 subunits are located in specific cell types in the retina. The subcellular segregation of the three subunits may underlie a physiological disparity in the spiking activity or modulation of different parts of the neuron. Future studies will investigate the functional roles of Kv3 channels in the retina
— id: 92528, year: 2001, vol: 27, page: 2148, stat: Journal Article,

Potassium channel-mediated presynaptic inhibition of GABAergic synaptic transmission in the mouse brain
Petit-Jacques, J.; Chang, S.; Ozaita, A.; Rudy, B.
2001 ;27(1):712-712, Abstracts (Society for Neuroscience)
Neurotransmitters regulate synaptic transmission by activating K+ channels (GIRK channels) and inhibiting Ca2+ channels via a membrane-delimited action of subunits of activated G proteins. Several examples of inhibition of synaptic transmission as a result of the activation of post-synaptic GIRK channels have been documented in the mammalian CNS. However, pre-synaptic inhibition initiated by metabotropic receptors is thought to be mediated mainly through the inhibition of Ca2+ channels, rather than by the activation of K+ channels. In fact the presence of presynaptic GIRK channels has remained controversial. We now present data suggesting that GIRK channels mediate presynaptic inhibition of GABA release. Utilizing whole cell recording methods, we measured spontaneous IPSC's in pyramidal neurons in acute slices. The recording pipette contained QX314 and Cs+ to block post-synaptic voltage-gated Na+ and K+ channels. QX314 also blocks GIRK channels. Bath application of tertiapin a drug known to block GIRK channels (as well as ROMK1 inward rectifier K+ channels) produced an increase in IPSC frequency without effect on mean amplitude. Similar effects were produced by bath application of 200 uM Ba2+, a concentration known to block GIRK and other inward rectifier K+ channels. Immunohistochemistry with antibodies to GIRK1 proteins demonstrated punctate staining surrounding cortical pyramidal neurons, suggestive of GIRK1 expression in the basket axo-somatic terminals of GABAergic interneurons. Together, the results suggest that presynaptic inward rectifier K+ channels, probably GIRK-type, can regulate GABAergic transmitter release
— id: 92530, year: 2001, vol: 27, page: 712, stat: Journal Article,

Kv3 channels: voltage-gated K+ channels designed for high-frequency repetitive firing
Rudy B; McBain CJ
2001 Sep;24(9):517-526, Trends in neurosciences
Analysis of the Kv3 subfamily of K(+) channel subunits has lead to the discovery of a new class of neuronal voltage-gated K(+) channels characterized by positively shifted voltage dependencies and very fast deactivation rates. These properties are adaptations that allow these channels to produce currents that can specifically enable fast repolarization of action potentials without compromising spike initiation or height. The short spike duration and the rapid deactivation of the Kv3 currents after spike repolarization maximize the quick recovery of resting conditions after an action potential. Several neurons in the mammalian CNS have incorporated into their repertoire of voltage-dependent conductances a relatively large number of Kv3 channels to enable repetitive firing at high frequencies - an ability that crucially depends on the special properties of Kv3 channels and their impact on excitability
— id: 26701, year: 2001, vol: 24, page: 517, stat: Journal Article,

Differential expression of genes encoding subthreshold-operating voltage-gated k+ channels in brain
Saganich MJ; Machado E; Rudy B
2001 Jul 1;21(13):4609-4624, Journal of neuroscience
The members of the three subfamilies (eag, erg, and elk) of the ether-a-go-go (EAG) family of potassium channel pore-forming subunits express currents that, like the M-current (I(M)), could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. A nonradioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the eight known EAG family subunits in rat brain was performed to identify neuronal populations in which the physiological roles of EAG channels could be studied. These distributions were compared with those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus; however, only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2, and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses
— id: 21165, year: 2001, vol: 21, page: 4609, stat: Journal Article,

Kt3.2 and kt3.3, two novel human two-pore k(+) channels closely related to task-1
Vega-Saenz De Miera E; Lau DH; Zhadina M; Pountney D; Coetzee WA; Rudy B
2001 Jul;86(1):130-142, Journal of neurophysiology
We report the cloning of human KT3.2 and KT3.3 new members of the two-pore K(+) channel (KT) family. Based on amino acid sequence and phylogenetic analysis, KT3.2, KT3.3, and TASK-1 constitute a subfamily within the KT channel mammalian family. When Xenopus oocytes were injected with KT3.2 cRNA, the resting membrane potential was brought close to the potassium equilibrium potential. At low extracellular K(+) concentrations, two-electrode voltage-clamp recordings revealed the expression of predominantly outward currents. With high extracellular K(+) (98 mM), the current-voltage relationship exhibited weak outward rectification. Measurement of reversal potentials at different [K(+)](o) revealed a slope of 48 mV per 10-fold change in K(+) concentration as expected for a K(+)-selective channel. Unlike TASK-1, which is highly sensitive to changes of pH in the physiological range, KT3.2 currents were relatively insensitive to changes in intracellular or extracellular pH within this range due to a shift in the pH dependency of KT3.2 of 1 pH unit in the acidic direction. On the other hand, the phorbol ester phorbol 12-myristate 13-acetate (PMA), which does not affect TASK-1, produces strong inhibition of KT3.2 currents. Human KT3.2 mRNA expression was most prevalent in the cerebellum. In rat, KT3.2 is exclusively expressed in the brain, but it has a wide distribution within this organ. High levels of expression were found in the cerebellum, medulla, and thalamic nuclei. The hippocampus has a nonhomogeneous distribution, expressing at highest levels in the lateral posterior and inferior portions. Medium expression levels were found in neocortex. The KT3.2 gene is located at chromosome 8q24 1-3, and the KT3.3 gene maps to chromosome 20q13.1
— id: 21157, year: 2001, vol: 86, page: 130, stat: Journal Article,

H2 histamine receptor-phosphorylation of Kv3.2 modulates interneuron fast spiking
Atzori M; Lau D; Tansey EP; Chow A; Ozaita A; Rudy B; McBain CJ
2000 Aug;3(8):791-798, Nature neuroscience
Histamine-containing neurons of the tuberomammilary nucleus project to the hippocampal formation to innervate H1 and H2 receptors on both principal and inhibitory interneurons. Here we show that H2 receptor activation negatively modulates outward currents through Kv3.2-containing potassium channels by a mechanism involving PKA phosphorylation in inhibitory interneurons. PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons, which in turn negatively modulated high-frequency population oscillations recorded in principal cell layers. All these effects were absent in a Kv3.2 knockout mouse. These data reveal a novel pathway for histamine-dependent regulation of high-frequency oscillations within the hippocampal formation
— id: 18829, year: 2000, vol: 3, page: 791, stat: Journal Article,

Histamine (H2) receptor modulation of Kv3.2 in fast-spiking interneurons
Atzori, M.; Phillips-Tansey, E.; Lau, D.; Ozaita, A.; Chow, A.; Rudy, B.; McBain, C. J.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
Histamine-containing neurons of the hypothalamic tuberomammilary nucleus project to the hippocampal formation where they innervate both principal and inhibitory interneurons (INs) via H1 and H2 receptors. H2 receptor activation is positively linked to adenylyl cyclase formation although the downstream targets of these receptors are unclear. Using whole-cell recording of K+ currents from Dentate Gyrus INs in mouse hippocampal slices we now show that H2 receptor activation decreases K+ currents through Kv3.2-containing channels by a mechanism involving PKA phosphorylation. Currents through Kv3.2 channels activate rapidly, are essentially non-inactivating and endow INs with a 'fast spiking' phenotype. Histamine (10 uM) or dimaprit (H2 agonist, 10 uM) application reduced the K+-current by 40+-4% (n=13), and 28+-5% (n=10) respectively. Incubation in either the H2 antagonist cimetidine or PKA inhibitors prevented the reduction of Kv3.2 current by H2 agonists. H2 receptor-dependent-PKA phosphorylation of Kv3.2 lowered the maximum firing frequency of inhibitory neurons (24+-3% decrease), which in turn decreased the high frequency (>70Hz) component of extracellular oscillations recorded in principal cell layers. All of these effects were reproduced by application of the PKA activator dibutyryl-cAMP (2mM) + IBMX (100 uM). H2 receptor modulation of outward currents and firing properties was absent in a Kv3.2 knockout mouse. Our data show that Kv3.2 containing channels are a target for histaminergic modulation and reveal a novel pathway for histamine-dependent regulation of high frequency oscillations within the hippocampal formation
— id: 92532, year: 2000, vol: 26, page: ?, stat: Journal Article,

Cloning of two novel human two-pore K+ channels closely related to TASK1
de Miera, ECVS; Pountney, D; Coetzee, W; Rudy, B
2000 JAN ;78(1):206A-206A, Biophysical journal
— id: 54762, year: 2000, vol: 78, page: 206A, stat: Journal Article,

Synaptic responses to whisker stimulation in mouse S1 barrel cortex in vivo
Harvey, M. A.; Wilent, W. B.; Rudy, B.; Contreras, D.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
We recorded intracellularly with sharp electrodes from layers 2 to 6 of S1 barrel cortex, in Ketamine-xylazine anesthetized mice in vivo. The bipolar EEG (pial surface - depth) was obtained simultaneously in the vicinity (usually apprx 1mm) of the intracellular pipette. Cells were identified as regular spiking, intrinsically bursting, fast spiking or regular fast bursting, according to the firing patterns in response to current injection through the micropipette and during synaptic activity evoked by thalamic or cortical stimulation. In addition corticothalamic projection cells were identified by their antidromic responses to thalamic stimulation. Cells oscillated in synchrony with the local EEG during the slow oscillation as described elsewhere (Steriade et al., 1993 J Neurosci. 13:3252-65; Contreras D, Steriade M 1995 J Neurosci 15: 604-22). In addition, most cells showed spontaneous spindle oscillations in synchrony with the local bipolar EEG. Cells responded with an epsp-ipsp sequence to weak electrical stimulation of the ventrobasal nucleus of the thalamus. These responses were very similar to stimulation of individual or multiple whiskers. Whiskers were stimulated with either a small hand held probe or with a piezoelectric device described by Simons, (Simons, D.J. 1983 Brain Res. 276: 178-182). In order to measure the contribution of ipsps to the responses to whisker stimulation we displaced cell's membrane potential with direct current and constructed current vs. voltage plots. In addition some cells (n=6) were recorded with pipettes filled with KCl in order to reverse chloride dependent ipsps
— id: 92537, year: 2000, vol: 26, page: ?, stat: Journal Article,

Impaired fast-spiking, suppressed cortical inhibition, and increased susceptibility to seizures in mice lacking Kv3.2 K+ channel proteins
Lau D; Vega-Saenz de Miera EC; Contreras D; Ozaita A; Harvey M; Chow A; Noebels JL; Paylor R; Morgan JI; Leonard CS; Rudy B
2000 Dec 15;20(24):9071-9085, Journal of neuroscience
Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 -/- mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing
— id: 18828, year: 2000, vol: 20, page: 9071, stat: Journal Article,

Altered firing properties of fast spiking and low-threshold spiking neurons in the neocortex of Kv3.2 knockout mice
Lau, D. H.; Petit-Jacques, J.; Rudy, B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
Inhibitory interneurons have critical functions in neocortical circuits. There are several types of inhibitory interneurons in the neocortex which differ in morphology, axonal connectivity, and firing patterns. Two major groups of inhibitory interneurons are the parvalbumin-containing fast spiking (FS) and the somatostatin-containing low-threshold spiking (LTS) cells. Both types are known to function in independent neuronal networks through reciprocal synaptic connections and gap junctions. Both types of neurons are known to contain Kv3 potassium channels. Kv3.2 and Kv3.1 channels are thought to be involved in the high frequency firing characteristic behavior of these cells due to their unusual channel properties. Kv3.2-Kv3.1 channels do not show significant activation until -10 mV and have the fastest deactivation rates of all cloned voltage-gated K+ channels. Using thalamocortical brain slices we studied the firing characteristics of FS and LTS cells in wild-type and Kv3.2 knockout (KO) mice. Electrophysiological properties of FS and LTS cells in deep neocortical layers were altered in the KO both in their repetitive firing properties and action potential waveforms. Steady state firing frequency of FS cells was lower in the KO than WT. In addition, KO FS cells were impaired in their ability to sustain high frequency firing. The cellular alterations of these interneurons may disrupt the synchrony of the inhibitory neuronal networks as suggested by in vivo data from Kv3.2 mutants
— id: 92536, year: 2000, vol: 26, page: ?, stat: Journal Article,

Presynaptic voltage-gated channel regulation by PYK2 tyrosine kinase
Mareno, H; Lev, S; Schlessinger, J; Rudy, B; Llinas, R
2000 NOV ;12(11):61-61, European journal of neuroscience
— id: 54453, year: 2000, vol: 12, page: 61, stat: Journal Article,

Frequenin, a Ca2+-binding protein, is expressed in heart and is a novel regulator of Kv4 currents
Nakamura, TY; Nadal, MS; Rudy, B; Artman, M; Coetzee, WA
2000 OCT 31 ;102(18):91-91, Circulation
— id: 55243, year: 2000, vol: 102, page: 91, stat: Journal Article,

Role of alternatively-spliced C-termini in the subcellular localization of Kv3 potassium channels in neurons
Ozaita, A.; Vega-Saenz de Miera, E.; Johns, D. C.; Marban, E.; Rudy, B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
The role of a given potassium channel in neuronal excitability depends on its subcellular localization within the neuron. Several K+ channel genes encode multiple alternatively spliced proteins which differ only in their C-terminal sequence and produce channels with similar electrophysiological properties. We have previously shown that alternatively spliced products of the Kv3.2 and Kv3.1 genes are differentially localized when expressed in a polarized system such as MDCK cells, and that Kv3.1 isoforms show a differential expression in mouse brain, suggesting that the intracellular C-terminal domain is responsible for the differential targeting. We have studied the distribution of the different isoforms in infected 10-12 d.i.v. hippocampal neurons obtained from Kv3.2 knockout mice. By using the ecdysone inducible expression system combined with adenoviral vectors we show that, as expected, Kv3.2a was in axons in few cases (10% stained axons, n=21) while Kv3.2b infected neurons showed 77% stained axons, n=36, at 3 hours after induction. At longer times, both isoforms showed a similar distribution (Kv3.2a: 60%, n=29; Kv3.2b: 56%, n=28 at 9h after induction). These results support the conclusion that Kv3 C-terminal domains dictate the differential targeting behavior. However, it appears that protein overexpression can 'overload' the targeting machinery resulting in ectopic localization
— id: 92535, year: 2000, vol: 26, page: ?, stat: Journal Article,

Expression and function of Eag and Kcnq K+ channels in brain
Saganich, M. J.; Machado, E.; Tyler, C.; Leonard, C.; Rudy, B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
M-currents are believed to play critical roles in the subthreshold behavior, and response to synaptic inputs of many CNS neurons. M-currents were first described in peripheral sympathetic neurons and have since been identified in neurons of the hippocampus, neocortex and olfactory bulb. Characteristic features of the classical M-current include a relatively low activation voltage, no measurable inactivation, and a low sensitivity to commonly used K+ channel blockers (4-AP and TEA). Another defining feature of the M-current is its inhibition by Ach via muscarinic receptors. It has been suggested that Kcnq2 and Kcnq3 subunits, when found in heteromultimers, are responsible for the classical M-current in sympathetic ganglia. However it is still unclear whether these genes are the molecular components for all M-like currents. Some members of the Eag family (eag,erg,elk) express voltage dependant currents with properties that resemble in some respects those of the M-current. These properties include a low voltage of activation and the ability to carry steady state currents in sub-threshold ranges. Eag K+ channels could thus play similar roles in neuronal excitability as members of the Kcnq family. To explore the role of these various genes in neuronal excitability, we used non-radioactive in situ hybridization to study the expression pattern of the Kcnq and Eag families of K+ channels in mouse and rat brain with particular emphasis on the thalamocortical system. These studies were then used to select cell types for electrophysiological recordings of M-like currents in vitro
— id: 92534, year: 2000, vol: 26, page: ?, stat: Journal Article,

Analysis of Kv4 chimeras reveals distinct roles for the N- and C-termini in the inactivation process
Sun, ZQ; Pountney, DJ; Ueda, S; Porter, L; Nakamura, TY; Rudy, B; Covarrubias, M; Artman, M; Coetzee, WA
2000 OCT 31 ;102(18):260-261, Circulation
— id: 55246, year: 2000, vol: 102, page: 260, stat: Journal Article,

Expression of two pore K+ channels in the CNS
Vega-Saenz de Miera, E.; Ozaita, A.; Zadina, M.; Rudy, B.
2000 ;26(1-2):?-?, Abstracts (Society for Neuroscience)
Several two pore K+ channels (KT) have been identified in mammals. Amino acid sequence and phylogenetic analysis suggest the existence of 8 distinct subfamilies, with 3 subfamilies containing at least two members. Eight KT genes are expressed in the CNS. In heterologous expression systems KT genes produce currents at resting membrane potentials and therefore, are likely to play important roles in neuronal subthreshold properties. The diversity of these channels may be associated with a high degree of cellular or subcellular specificity. The diversity also provides multiple ways to regulate the subthreshold behavior of neurons. In situ hybridization experiments revealed that TASK-1 and KT3.2 are co-expressed in cerebellar granule cell layer, the olfactory bulb and in several brainstem nuclei like the ambigual, motor trigeminal, facial, vagal and hypoglossal nuclei. These two channels belong to the same subfamily (75% identity and 86% similarity in the core region) but differ in their pH dependence, and modulation by second messengers. TASK-1 is very sensitive to changes in pH in the physiological range while KT3.2 is almost insensitive to pH changes in the pH 7.0 to 8.0 range. KT3.2 is modulated by the phorbol ester PMA, a potent protein kinase C stimulator, but TASK-1 is insensitive to this treatment. If these channels are expressed in the same cells they may form heteromultimeric channels. Therefore the ability to form heteromeric complexes in vitro was investigated
— id: 92533, year: 2000, vol: 26, page: ?, stat: Journal Article,

PKA phosphorylation of Kv3.2 modulates high frequency firing in hippocampal interneurons
Atzori, M.; Phillips-Tansey, E.; Lau, D.; Ozaita, A.; Chow, A.; Rudy, B.; McBain, C. J.
1999 ;25(1-2):455-455, Abstracts (Society for Neuroscience)
— id: 92541, year: 1999, vol: 25, page: 455, stat: Journal Article,

FGF-2 potentiates Ca(2+)-dependent inactivation of NMDA receptor currents in hippocampal neurons
Boxer AL; Moreno H; Rudy B; Ziff EB
1999 Dec;82(6):3367-3377, Journal of neurophysiology
Peptide growth factors such as the neurotrophins and fibroblast growth factors have potent effects on synaptic transmission, development, and cell survival. We report that chronic (hours) treatment with basic fibroblast growth factor (FGF-2) potentiates Ca(2+)-dependent N-methyl-D-aspartate (NMDA) receptor inactivation in cultured hippocampal neurons. This effect is specific for the NMDA-subtype of ionotropic glutamate receptor and FGF-2. The potentiated inactivation requires ongoing protein synthesis during growth factor treatment and the activity of protein phosphatase 2B (PP2B or calcineurin) during agonist application. These results suggest a mechanism by which FGF-2 receptor signaling may regulate neuronal survival and synaptic plasticity
— id: 11900, year: 1999, vol: 82, page: 3367, stat: Journal Article,

Immunocytochemical evidence for Kv3.1b K+ channel subunits in laterodorsal (LDT) and pedunculopontine (PPT) tegmental nuclei in mouse
Burlet, S.; Tyler, C. J.; Chow, A.; Joho, R. H.; Lau, D.; Rudy, B.; Leonard, C. S.
1999 ;25(1-2):2145-2145, Abstracts (Society for Neuroscience)
— id: 92539, year: 1999, vol: 25, page: 2145, stat: Journal Article,

K(+) channel expression distinguishes subpopulations of parvalbumin- and somatostatin-containing neocortical interneurons
Chow A; Erisir A; Farb C; Nadal MS; Ozaita A; Lau D; Welker E; Rudy B
1999 Nov 1;19(21):9332-9345, Journal of neuroscience
Kv3.1 and Kv3.2 K(+) channel proteins form similar voltage-gated K(+) channels with unusual properties, including fast activation at voltages positive to -10 mV and very fast deactivation rates. These properties are thought to facilitate sustained high-frequency firing. Kv3.1 subunits are specifically found in fast-spiking, parvalbumin (PV)-containing cortical interneurons, and recent studies have provided support for a crucial role in the generation of the fast-spiking phenotype. Kv3.2 mRNAs are also found in a small subset of neocortical neurons, although the distribution of these neurons is different. We raised antibodies directed against Kv3.2 proteins and used dual-labeling methods to identify the neocortical neurons expressing Kv3.2 proteins and to determine their subcellular localization. Kv3.2 proteins are prominently expressed in patches in somatic and proximal dendritic membrane as well as in axons and presynaptic terminals of GABAergic interneurons. Kv3.2 subunits are found in all PV-containing neurons in deep cortical layers where they probably form heteromultimeric channels with Kv3.1 subunits. In contrast, in superficial layer PV-positive neurons Kv3.2 immunoreactivity is low, but Kv3.1 is still prominently expressed. Because Kv3.1 and Kv3.2 channels are differentially modulated by protein kinases, these results raise the possibility that the fast-spiking properties of superficial- and deep-layer PV neurons are differentially regulated by neuromodulators. Interestingly, Kv3. 2 but not Kv3.1 proteins are also prominent in a subset of seemingly non-fast-spiking, somatostatin- and calbindin-containing interneurons, suggesting that the Kv3.1-Kv3.2 current type can have functions other than facilitating high-frequency firing
— id: 11941, year: 1999, vol: 19, page: 9332, stat: Journal Article,

Molecular diversity of K+ channels
Coetzee WA; Amarillo Y; Chiu J; Chow A; Lau D; McCormack T; Moreno H; Nadal MS; Ozaita A; Pountney D; Saganich M; Vega-Saenz de Miera E; Rudy B
1999 Apr 30;868:233-285, Annals of the New York Academy of Sciences
K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates
— id: 11979, year: 1999, vol: 868, page: 233, stat: Journal Article,

Function of specific K(+) channels in sustained high-frequency firing of fast-spiking neocortical interneurons
Erisir A; Lau D; Rudy B; Leonard CS
1999 Nov;82(5):2476-2489, Journal of neurophysiology
Fast-spiking GABAergic interneurons of the neocortex and hippocampus fire high-frequency trains of brief action potentials with little spike-frequency adaptation. How these striking properties arise is unclear, although recent evidence suggests K(+) channels containing Kv3.1-Kv3.2 proteins play an important role. We investigated the role of these channels in the firing properties of fast-spiking neocortical interneurons from mouse somatosensory cortex using a pharmacological and modeling approach. Low tetraethylammonium (TEA) concentrations (</=1 mM), which block only a few known K(+) channels including Kv3.1-Kv3.2, profoundly impaired action potential repolarization and high-frequency firing. Analysis of the spike trains evoked by steady depolarization revealed that, although TEA had little effect on the initial firing rate, it strongly reduced firing frequency later in the trains. These effects appeared to be specific to Kv3.1 and Kv3.2 channels, because blockade of dendrotoxin-sensitive Kv1 channels and BK Ca(2+)-activated K(+) channels, which also have high TEA sensitivity, produced opposite or no effects. Voltage-clamp experiments confirmed the presence of a Kv3.1-Kv3.2-like current in fast-spiking neurons, but not in other interneurons. Analysis of spike shape changes during the spike trains suggested that Na(+) channel inactivation plays a significant role in the firing-rate slowdown produced by TEA, a conclusion that was supported by computer simulations. These findings indicate that the unique properties of Kv3.1-Kv3.2 channels enable sustained high-frequency firing by facilitating the recovery of Na(+) channel inactivation and by minimizing the duration of the afterhyperpolarization in neocortical interneurons
— id: 32242, year: 1999, vol: 82, page: 2476, stat: Journal Article,

Kv3.1-Kv3.2 channels underlie a high-voltage-activating component of the delayed rectifier K+ current in projecting neurons from the globus pallidus
Hernandez-Pineda R; Chow A; Amarillo Y; Moreno H; Saganich M; de Miera EV; Hernandez-Cruz A; Rudy B
1999 Sep;82(3):1512-1528, Journal of neurophysiology
The globus pallidus plays central roles in the basal ganglia circuitry involved in movement control as well as in cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus. Most pallidal neurons are GABAergic projecting cells, a large fraction of which express the calcium binding protein parvalbumin (PV). Here we show that PV-containing pallidal neurons coexpress Kv3. 1 and Kv3.2 K+ channel proteins and that both Kv3.1 and Kv3.2 antibodies coprecipitate both channel proteins from pallidal membrane extracts solubilized with nondenaturing detergents, suggesting that the two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2 channels have several unusual electrophysiological properties when expressed in heterologous expression systems and are thought to play special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in the cortex and the hippocampus. Electrophysiological analysis of freshly dissociated pallidal neurons demonstrates that these cells have a current that is nearly identical to the currents expressed by Kv3.1 and Kv3.2 proteins in heterologous expression systems, including activation at very depolarized membrane potentials (more positive than -10 mV) and very fast deactivation rates. These results suggest that the electrophysiological properties of native channels containing Kv3.1 and Kv3.2 proteins in pallidal neurons are not significantly affected by factors such as associated subunits or postranslational modifications that result in channels having different properties in heterologous expression systems and native neurons. Most neurons in the globus pallidus have been reported to fire sustained trains of action potentials at high-frequency. Kv3.1-Kv3.2 voltage-gated K+ channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons
— id: 8478, year: 1999, vol: 82, page: 1512, stat: Journal Article,

Sensory inputs modulate slow EEG rhythms in the anesthetized mice
Lau, D. H. P.; Contreras, D.; Rudy, B.
1999 ;25(1-2):2194-2194, Abstracts (Society for Neuroscience)
— id: 92538, year: 1999, vol: 25, page: 2194, stat: Journal Article,

The effects of Shaker beta-subunits on the human lymphocyte K+ channel Kv1.3
McCormack T; McCormack K; Nadal MS; Vieira E; Ozaita A; Rudy B
1999 Jul 16;274(29):20123-20126, Journal of biological chemistry
The activation of T-lymphocytes is dependent upon, and accompanied by, an increase in voltage-gated K+ conductance. Kv1.3, a Shaker family K+ channel protein, appears to play an essential role in the activation of peripheral human T cells. Although Kv1.3-mediated K+ currents increase markedly during the activation process in mice, and to a lesser degree in humans, Kv1.3 mRNA levels in these organisms do not, indicating post-transcriptional regulation. In other tissues Shaker K+ channel proteins physically associate with cytoplasmic beta-subunits (Kvbeta1-3). Recently it has been shown that Kvbeta1 and Kvbeta2 are expressed in mouse T cells and that they are up-regulated during mitogen-stimulated activation. In this study, we show that the human Kvbeta subunits substantially increase K+ current amplitudes when coexpressed with their Kv1.3 counterpart, and that unlike in mouse, protein levels of human Kvbeta2 remain constant upon activation. Differences in Kvbeta2 expression between mice and humans may explain the differential K+ conductance increases which accompany T-cell proliferation in these organisms
— id: 11991, year: 1999, vol: 274, page: 20123, stat: Journal Article,

Chromosomal mapping of the potassium channel genes Kcnq2 and Kcnq3 in mouse
McCormack T; Rudy B; Seldin MF
1999 Mar 15;56(3):360-361, Genomics
— id: 6068, year: 1999, vol: 56, page: 360, stat: Journal Article,

The role of Kir2.1 in the genesis of native cardiac inward-rectifier K+ currents during pre- and postnatal development
Nakamura TY; Lee K; Artman M; Rudy B; Coetzee WA
1999 Apr 30;868:434-437, Annals of the New York Academy of Sciences
Our results demonstrate that (a) the Kir2.1 gene encodes a native K+ channel protein with a 21-pS conductance; (b) this channel has an important role in the genesis of adult ventricular 1K1; and (c) the contribution of Kir2.1 channel proteins to 1K1 changes during development. The lack of contribution of Kir2.1 to fetal 1K1 channels is interesting from the point of view of possible future generation of knockout mice lacking Kir2.1, since cardiac abnormalities would not be expected to result in fetal lethality. These observations provide further support for a generalized hypothesis that different genes may code for 1K1 channel proteins at various developmental stages. However, the effects of these AS-oligos must first be examined on native 1K1 channels in cardiac myocytes before definite conclusions can be reached
— id: 6164, year: 1999, vol: 868, page: 434, stat: Journal Article,

Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K+ channel principal subunit
Pountney DJ; Gulkarov I; Vega-Saenz de Miera E; Holmes D; Saganich M; Rudy B; Artman M; Coetzee WA
1999 May 7;450(3):191-196, FEBS letters
We have identified and cloned a new member of the mammalian tandem pore domain K+ channel subunit family, TWIK-originated similarity sequence, from a human testis cDNA library. The 939 bp open reading frame encodes a 313 amino acid polypeptide with a calculated Mr of 33.7 kDa. Despite the same predicted topology, there is a relatively low sequence homology between TWIK-originated similarity sequence and other members of the mammalian tandem pore domain K+ channel subunit family group. TWIK-originated similarity sequence shares a low (< 30%) identity with the other mammalian tandem pore domain K+ channel subunit family group members and the highest identity (34%) with TWIK-1 at the amino acid level. Similar low levels of sequence homology exist between all members of the mammalian tandem pore domain K+ channel subunit family. Potential glycosylation and consensus PKC sites are present. Northern analysis revealed species and tissue-specific expression patterns. Expression of TWIK-originated similarity sequence is restricted to human pancreas, placenta and heart, while in the mouse, TWIK-originated similarity sequence is expressed in the liver. No functional currents were observed in Xenopus laevis oocytes or HEK293T cells, suggesting that TWIK-originated similarity sequence may be targeted to locations other than the plasma membrane or that TWIK-originated similarity sequence may represent a novel regulatory mammalian tandem pore domain K+ channel subunit family subunit
— id: 8510, year: 1999, vol: 450, page: 191, stat: Journal Article,

Opposite effects of PKC activation on Kv4.1 and Kv4.2 currents
Pountney, David J; Covarrubias, Manuel L; Nakamura, Tomoe Y; Porter, Lisa M; Artman, Michael; Rudy, Bernardo; Coetzee, William A
1999 Nov 7-10;110(18 SUPPL.):I63-I63, Circulation
— id: 15878, year: 1999, vol: 110, page: I63, stat: Journal Article,

Apposite effects of PKC activation on kv4.1 and kv4.2 currents
Pountney, DJ; Covarrubias, ML; Jefferson, T; Nakamura, TY; Porter, LM; Artman, M; Rudy, B; Coetzee, WA
1999 NOV 2 ;100(18):63-63, Circulation
— id: 53786, year: 1999, vol: 100, page: 63, stat: Journal Article,

Molecular diversity of ion channels and cell function
Rudy B
1999 Apr 30;868:1-12, Annals of the New York Academy of Sciences
— id: 11980, year: 1999, vol: 868, page: 1, stat: Journal Article,

Contributions of Kv3 channels to neuronal excitability
Rudy B; Chow A; Lau D; Amarillo Y; Ozaita A; Saganich M; Moreno H; Nadal MS; Hernandez-Pineda R; Hernandez-Cruz A; Erisir A; Leonard C; Vega-Saenz de Miera E
1999 Apr 30;868:304-343, Annals of the New York Academy of Sciences
Four mammalian Kv3 genes have been identified, each of which generates, by alternative splicing, multiple protein products differing in their C-terminal sequence. Products of the Kv3.1 and Kv3.2 genes express similar delayed-rectifier type currents in heterologous expression systems, while Kv3.3 and Kv3.4 proteins express A-type currents. All Kv3 currents activate relatively fast at voltages more positive than -10 mV, and deactivate very fast. The distribution of Kv3 mRNAs in the rodent CNS was studied by in situ hybridization, and the localization of Kv3.1 and Kv3.2 proteins has been studied by immunohistochemistry. Most Kv3.2 mRNAs (approximately 90%) are present in thalamic-relay neurons throughout the dorsal thalamus. The protein is expressed mainly in the axons and terminals of these neurons. Kv3.2 channels are thought to be important for thalamocortical signal transmission. Kv3.1 and Kv3.2 proteins are coexpressed in some neuronal populations such as in fast-spiking interneurons of the cortex and hippocampus, and neurons in the globus pallidus. Coprecipitation studies suggest that in these cells the two types of protein form heteromeric channels. Kv3 proteins appear to mediate, in native neurons, similar currents to those seen in heterologous expression systems. The activation voltage and fast deactivation rates are believed to allow these channels to help repolarize action potentials fast without affecting the threshold for action potential generation. The fast deactivating current generates a quickly recovering after hyperpolarization, thus maximizing the rate of recovery of Na+ channel inactivation without contributing to an increase in the duration of the refractory period. These properties are believed to contribute to the ability of neurons to fire at high frequencies and to help regulate the fidelity of synaptic transmission. Experimental evidence has now become available showing that Kv3.1-Kv3.2 channels play critical roles in the generation of fast-spiking properties in cortical GABAergic interneurons
— id: 11978, year: 1999, vol: 868, page: 304, stat: Journal Article,

Molecular and functional diversity of ion channels and receptors
Rudy, Bernardo; Seeburg, P. H
New York : New York Academy of Sciences, 1999,
— id: 610, year: 1999, vol: , page: , stat: ,

Cloning of components of a novel subthreshold-activating K(+) channel with a unique pattern of expression in the cerebral cortex
Saganich MJ; Vega-Saenz de Miera E; Nadal MS; Baker H; Coetzee WA; Rudy B
1999 Dec 15;19(24):10789-10802, Journal of neuroscience
Potassium channels that are open at very negative membrane potentials govern the subthreshold behavior of neurons. These channels contribute to the resting potential and help regulate the degree of excitability of a neuron by affecting the impact of synaptic inputs and the threshold for action potential generation. They can have large influences on cell behavior even when present at low concentrations because few conductances are active at these voltages. We report the identification of a new K(+) channel pore-forming subunit of the ether-a-go-go (Eag) family, named Eag2, that expresses voltage-gated K(+) channels that have significant activation at voltages around -100 mV. Eag2 expresses outward-rectifying, non-inactivating voltage-dependent K(+) currents resembling those of Eag1, including a strong dependence of activation kinetics on prepulse potential. However, Eag2 currents start activating at subthreshold potentials that are 40-50 mV more negative than those reported for Eag1. Because they activate at such negative voltages and do not inactivate, Eag2 channels will contribute sustained outward currents down to the most negative membrane potentials known in neurons. Although Eag2 mRNA levels in whole brain appear to be low, they are highly concentrated in a few neuronal populations, most prominently in layer IV of the cerebral cortex. This highly restricted pattern of cortical expression is unlike that of any other potassium channel cloned to date and may indicate specific roles for this channel in cortical processing. Layer IV neurons are the main recipient of the thalamocortical input. Given their functional properties and specific distribution, Eag2 channels may play roles in the regulation of the behavioral state-dependent entry of sensory information to the cerebral cortex
— id: 8599, year: 1999, vol: 19, page: 10789, stat: Journal Article,

Cloning of a new eag potassium subunit expressed primarily in layer IV of the rat neocortex
Saganich, M.; Vega-Saenz de Miera, E.; Nadal, M.; Chow, A.; Baker, H.; Rudy, B.
1999 ;25(1-2):691-691, Abstracts (Society for Neuroscience)
— id: 92540, year: 1999, vol: 25, page: 691, stat: Journal Article,

Distribution of the potassium channel subunit KV3.2 in rat cerebellum
Bobik, M.; Martone, M. E.; Chow, A.; Rudy, B.; Ellisman, M.
1998 ;24(1-2):1578-1578, Abstracts (Society for Neuroscience)
— id: 92544, year: 1998, vol: 24, page: 1578, stat: Journal Article,

KV3.1 and KV3.2 proteins distinguish three subpopulations of GABA-ergic interneurons in the mouse cortex
Chow, A.; Erisir, A.; Farb, C.; Lau, D. H. P.; Rudy, B.
1998 ;24(1-2):1579-1579, Abstracts (Society for Neuroscience)
— id: 92543, year: 1998, vol: 24, page: 1579, stat: Journal Article,

Low tea concentration disrupts high frequency firing of fast spiking cells in mouse somatosensory cortex
Erisir, A.; Lau, D.; Rudy, B.; Leonard, C. S.
1998 ;24(1-2):632-632, Abstracts (Society for Neuroscience)
— id: 92546, year: 1998, vol: 24, page: 632, stat: Journal Article,

Targeted disruption of a K+ channel gene that is principally expressed in the presynaptic terminals of thalamic relay neurons in mouse
Lau, D. H. P.; Castro-Alamancos, M.; Chow, A.; Ozaita, A.; Vega-Saenz De Miera, E.; Mathew, S.; Gibson, J.; Connors, B. W.; Rudy, B.
1998 ;24(1-2):128-128, Abstracts (Society for Neuroscience)
— id: 92547, year: 1998, vol: 24, page: 128, stat: Journal Article,

Isoform-specific modulation of rat Kv3 potassium channel splice variants
McIntosh, P; Moreno, H; Robertson, B; Rudy, B
1998 SEP ;511P(11):147P-147P, Journal of physiology
— id: 53688, year: 1998, vol: 511P, page: 147P, stat: Journal Article,

Calcium dependent activation of P type calcium channel by PYK2 mediated phosphorylation of an auxiliary subunit
Moreno, H.; Lev, S.; Hernandez, J.; Schlessinger, J.; Rudy, B.; Llinas, R.
1998 ;24(1-2):1577-1577, Abstracts (Society for Neuroscience)
— id: 92323, year: 1998, vol: 24, page: 1577, stat: Journal Article,

Inhibition of rat ventricular IK1 with antisense oligonucleotides targeted to Kir2.1 mRNA
Nakamura TY; Artman M; Rudy B; Coetzee WA
1998 Mar;274(3 Pt 2):H892-H900, American journal of physiology. Heart & circulatory physiology
The cardiac inward rectifying K+ current (IK1) is important in maintaining the maximum diastolic potential. We used antisense oligonucleotides to determine the role of Kir2.1 channel proteins in the genesis of native rat ventricular IK1. A combination of two antisense phosphorothioate oligonucleotides inhibited heterologously expressed Kir2.1 currents in Xenopus oocytes, either when coinjected with Kir2.1 cRNA or when applied in the incubation medium. Specificity was demonstrated by the lack of inhibition of Kir2.2 and Kir2.3 currents in oocytes. In rat ventricular myocytes (4-5 days culture), these oligonucleotides caused a significant reduction of whole cell IK1 (without reducing the transient outward K+ current or the L-type Ca2+ current). Cell-attached patches demonstrated the occurrence of multiple channel events in control myocytes (8, 14, 21, 35, 43, and 80 pS). The 21-pS channel was specifically knocked down in antisense-treated myocytes (fewer patches contained this channel, and its open frequency was reduced). These results demonstrate that the Kir2.1 gene encodes a specific native 21-pS K(+)-channel protein and that this channel has an essential role in the genesis of cardiac IK1
— id: 7702, year: 1998, vol: 274, page: H892, stat: Journal Article,

Inhibition of adult rat ventricular I-K1 with antisense oligonucleotides targeted to Kir2.1 mRNA
Nakamura, TY; Artman, M; Rudy, B; Coetzee, WA
1998 FEB ;74(2):A157-A157, Biophysical journal
— id: 53437, year: 1998, vol: 74, page: A157, stat: Journal Article,

Differential targeting of KV3.1-KV3.2 containing potassium channels produced by alternatively-spliced C-terminal
Ozaita, A.; Vega-Saez De Miera, E.; Chow, A.; Muth, T. R.; Caplan, M. J.; Rudy, B.
1998 ;24(1-2):1580-1580, Abstracts (Society for Neuroscience)
— id: 92542, year: 1998, vol: 24, page: 1580, stat: Journal Article,

Identification of currents mediated by channels with Kv3 subfamily proteins in neurons from the rat globus pallidus
Pineda, R.; Saganich, M.; Moreno, H.; Vega-Sanez De Miera, E.; Hernandez-Cruz, A.; Rudy, B.
1998 ;24(1-2):1330-1330, Abstracts (Society for Neuroscience)
— id: 92545, year: 1998, vol: 24, page: 1330, stat: Journal Article,

Differential expression of Kv4 K+ channel subunits mediating subthreshold transient K+ (A-type) currents in rat brain
Serodio P; Rudy B
1998 Feb;79(2):1081-1091, Journal of neurophysiology
The mammalian Kv4 gene subfamily and its Drosophila Shal counterpart encode proteins that form fast inactivating K+ channels that activate and inactivate at subthreshold potentials and recover from inactivation at a faster rate than other inactivating Kv channels. Taken together, the properties of Kv4 channels compare best with those of low-voltage activating 'A-currents' present in the neuronal somatodendritic compartment and widely reported across several types of central and peripheral neurons, as well as the (Ca2+-independent) transient outward potassium conductance of heart cells (Ito). Three distinct genes have been identified that encode mammalian Shal homologs (Kv4. 1, Kv4.2, and Kv4.3), of which the latter two are abundant in rat adult brain and heart tissues. The distribution in the adult rat brain of the mRNA transcripts encoding the three known Kv4 subunits was studied by in situ hybridization histochemistry. Kv4.1 signals are very faint, suggesting that Kv4.1 mRNAs are expressed at very low levels, but Kv4.2 and Kv4.3 transcripts appear to be abundant and each produces a unique pattern of expression. Although there is overlap expression of Kv4.2 and Kv4.3 transcripts in several neuronal populations, the dominant feature is one of differential, and sometimes reciprocal expression. For example, Kv4.2 transcripts are the predominant form in the caudate-putamen, pontine nucleus and several nuclei in the medula, whereas the substantia nigra pars compacta, the restrosplenial cortex, the superior colliculus, the raphe, and the amygdala express mainly Kv4.3. Some brain structures contain both Kv4.2 and Kv4.3 mRNAs but each dominates in distinct neuronal subpopulations. For example, in the olfactory bulb Kv4.2 dominates in granule cells and Kv4.3 in periglomerular cells. In the hippocampus Kv4.2 is the most abundant isoform in CA1 pyramidal cells, whereas only Kv4.3 is expressed in interneurons. Both are abundant in CA2-CA3 pyramidal cells and in granule cells of the dentate gyrus, which also express Kv4.1. In the dorsal thalamus strong Kv4.3 signals are seen in several lateral nuclei, whereas medial nuclei express Kv4.2 and Kv4.3 at moderate to low levels. In the cerebellum Kv4.3, but not Kv4.2, is expressed in Purkinje cells and molecular layer interneurons. In the cerebellar granule cell layer, the reciprocity between Kv4.2 and Kv4.3 is observed in subregions of the same neuronal population. In fact, the distribution of Kv4 channel transcripts in the cerebellum defines a new pattern of compartmentation of the cerebellar cortex and the first one involving molecules directly involved in signal processing
— id: 12138, year: 1998, vol: 79, page: 1081, stat: Journal Article,

Molecular characterization of the sodium channel subunits expressed in mammalian cerebellar Purkinje cells
de Miera EVS; Rudy B; Sugimori M; Llinas R
1997 Jun 24;94(13):7059-7064, Proceedings of the National Academy of Sciences of the United States of America
Inactivating and noninactivating Na+ conductances are known to generate, respectively, the rising phase and the prolonged plateau phase of cerebellar Purkinje cell (PC) action potentials. These conductances have different voltage activation levels, suggesting the possibility that two distinct types of ion channels are involved. Single Purkinje cell reverse transcription-PCR from guinea pig cerebellar slices identified two Na+ channel alpha subunit transcripts, the orthologs of RBI (rat brain I) and Nach6/Scn8a. The latter we shall name CerIII. In situ hybridization histochemistry in rat brain demonstrated broad CerIII expression at high levels in many neuronal groups in the brain and spinal cord, with little if any expression in white matter, or nerve tracts. RBII (rat brain II), the most commonly studied recombinant Na+ channel alpha subunit is not expressed in PCs. As the absence of Scn8a has been correlated with motor endplate disease (med), in which transient sodium currents are spared, RBI appears to be responsible for the transient sodium current in PC. Conversely, jolting mice with a mutated Scn8a message demonstrates PC abnormalities in rapid, simple spike generation, linking CerIII to the persistent sodium current
— id: 8020, year: 1997, vol: 94, page: 7059, stat: Journal Article,

beta subunits influence the biophysical and pharmacological differences between P- and Q-type calcium currents expressed in a mammalian cell line [published erratum appears in Proc Natl Acad Sci U S A 1998 Mar 3;95(5):2714]
Moreno H; Rudy B; Llinas R
1997 Dec 9;94(25):14042-14047, Proceedings of the National Academy of Sciences of the United States of America
Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit
— id: 9880, year: 1997, vol: 94, page: 14042, stat: Journal Article,

Electrophysiological and pharmacological characterization of recombinant putative P/Q type calcium channels in HEK 293 cells
Moreno, H.; Rudy, B.; Llinas, R.
1997 ;23(1-2):1193-1193, Abstracts (Society for Neuroscience)
— id: 92330, year: 1997, vol: 23, page: 1193, stat: Journal Article,

Modulation of Kv4 channels, key components of rat ventricular transient outward K+ current, by PKC
Nakamura TY; Coetzee WA; Vega-Saenz De Miera E; Artman M; Rudy B
1997 Oct;273(4 Pt 2):H1775-H1786, American journal of physiology. Heart & circulatory physiology
Current evidence suggests that members of the Kv4 subfamily may encode native cardiac transient outward current (I(to)). Antisense hybrid-arrest with oligonucleotides targeted to Kv4 mRNAs specifically inhibited rat ventricular I(to), supporting this hypothesis. To determine whether protein kinase C (PKC) affects I(to) by an action on these molecular components, we compared the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and rat ventricular I(to). Phorbol 12-myristate 13-acetate (PMA) suppressed both Kv4.2 and Kv4.3 currents as well as native I(to), but not after preincubation with PKC inhibitors (e.g., chelerythrine). An inactive stereoisomer of PMA had no effect. Phenylephrine or carbachol inhibited Kv4 currents only when coexpressed, respectively, with alpha1C-adrenergic or M1 muscarinic receptors (this inhibition was also prevented by chelerythrine). The voltage dependence and inactivation kinetics of Kv4.2 were unchanged by PKC, but small effects on the rates of inactivation and recovery from inactivation of native I(to) were observed. Thus Kv4.2 and Kv4.3 proteins are important subunits of native rat ventricular I(to), and PKC appears to reduce this current by affecting the molecular components of the channels mediating I(to)
— id: 12230, year: 1997, vol: 273, page: H1775, stat: Journal Article,

Kir2.1 antisense oligonucleotides decrease inward rectifier K+ currents expressed in Xenopus oocytes by poly(A(+)) RNA from neonatal and adult, but not fetal mouse ventricles
Nakamura, TY; Artman, M; Rudy, B; Coetzee, WA
1997 OCT 21 ;96(8):2380-2380, Circulation
— id: 105048, year: 1997, vol: 96, page: 2380, stat: Journal Article,

Modulation of transient K+ currents (Kv4.2 and Kv4.3) by protein kinase C
Nakamura, TY; Coetzee, WA; Vega, E; Artman, M; Rudy, B
1997 FEB ;72(2):TUP28-TUP28, Biophysical journal
— id: 53307, year: 1997, vol: 72, page: TUP28, stat: Journal Article,

K+ channel subunit isoforms with divergent carboxy-terminal sequences carry distinct membrane targeting signals
Ponce A; Vega-Saenz de Miera E; Kentros C; Moreno H; Thornhill B; Rudy B
1997 Sep 15;159(2):149-159, Journal of membrane biology
Kv3 K+ channel genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K+ channel proteins that differ only in their C-termini. This divergence does not affect the electrophysiological properties of the channels expressed by these proteins. A similar alternative splicing with unknown function is seen in K+ channel genes of other families. We have investigated the possibility that the alternative splicing serves to generate channel subunits with different membrane targeting signals by examining the sorting behavior of three alternatively-spliced Kv3.2 isoforms when expressed in polarized MDCK cells. Two Kv3.2 proteins, Kv3.2b and Kv3.2c were expressed predominantly in the apical membrane, while Kv3.2a was localized mainly to the basolateral side (thought to be equivalent to the axonal and somatodendritic compartments in neurons, respectively). The Kv3.2 mRNA transcripts used in these studies are identical except for their 3' sequence, encoding the extreme C-terminal domain of the protein and the 3'UTR of the mRNA. However, the proteins achieve the same localizations in MDCK cells when expressed from constructs containing or lacking the 3'UTR, indicating that the differential localization is due to targeting signals present in the C' terminal domain of the protein. These results suggest that the alternative splicing of Kv3 genes is involved in channel localization. Since the precise localization of any given ion channel on the neuronal surface has significant functional implications, the results shown here suggest an important function for the alternative splicing of 3' ends seen in many K+ channel genes
— id: 7236, year: 1997, vol: 159, page: 149, stat: Journal Article,

Subcellular localization of the K+ channel subunit Kv3.1b in selected rat CNS neurons
Sekirnjak C; Martone ME; Weiser M; Deerinck T; Bueno E; Rudy B; Ellisman M
1997 Aug 22;766(1-2):173-187, Brain research
Voltage-gated potassium channels constitute the largest group of heteromeric ion channels discovered to date. Over 20 genes have been isolated, encoding different channel subunit proteins which form functional tetrameric K+ channels. We have analyzed the subcellular localization of subunit Kv3.1b, a member of the Kv3 (Shaw-like) subfamily, in rat brain at the light and electron microscopic level, using immunocytochemical detection. Detailed localization was carried out in specific neurons of the neocortex, hippocampus and cerebellum. The identity of Kv3.1b-positive neurons was established using double labeling with markers for specific neuronal populations. In the neocortex, the Kv3.1b subunit was expressed in most parvalbumin-containing bipolar, basket or chandelier cells, and in some bipolar or double bouquet neurons containing calbindin. In the hippocampus, Kv3.1b was expressed in many parvalbumin-containing basket cells, as well as in calbindin-positive neurons in the stratum oriens, and in a small number of interneurons that did not stain for either parvalbumin or calbindin. Kv3.1b protein was not present in pyramidal cells in the neocortex and the hippocampus, but these cells were outlined by labeled presynaptic terminals from interneuron axons that surround the postsynaptic cell. In the cerebellar cortex, granule cells were the only population expressing the channel protein. Careful examination of individual granule cells revealed a non-uniform distribution of Kv3.1 staining on the somata: circular bands of labeling were present in the vicinity of the axon hillock. In cortical and hippocampal interneurons, as well as in cerebellar granule cells, the Kv3.1b subunit was present in somatic and unmyelinated axonal membranes and adjacent cytoplasm, as well as in the most proximal portion of dendritic processes, but not throughout most of the dendrite. Labeling was also seen in the terminals of labeled axons, but not at a higher concentration than in other parts of the axon. The distribution in the cells analyzed supports a role in action potential transmission by regulating action potential duration
— id: 18830, year: 1997, vol: 766, page: 173, stat: Journal Article,

Intrinsic Xenopus oocytes inward rectifiers are modulated by changes in redox potential
Amarillo, Y; Nakamura, TY; Oliveros, W; Coetzee, W; Rudy, B; Moreno, H
1996 FEB ;70(2):SU239-SU239, Biophysical journal
— id: 53047, year: 1996, vol: 70, page: SU239, stat: Journal Article,

Heterologous desensitization of the human endothelin A and neurokinin A receptors in Xenopus laevis oocytes
Cyr CR; Devi LA; Rudy B; Kris RM
1996 ;6(2):99-109, Receptors & signal transduction
Endothelin 1 (ET1) desensitizes endothelin A receptor for 90-110 min while neurokinin A (NKA) desensitizes neurokinin A receptor for 25-35 min in Xenopus laevis oocytes. In the present study, endothelin A receptor and neurokinin A receptor were coexpressed in Xenopus laevis oocytes in an effort to characterize heterologous desensitization of the receptors that activate phospholipase C-beta. ET1 desensitizes both the endothelin A receptor and the neurokinin A receptor for 90-110 min, whereas stimulation with NKA desensitizes the same two receptors for only 25-35 min. Homologous and heterologous desensitization experiments were also carried out with endothelin 3 (ET3), a ligand that exhibits lower affinity to the endothelin A receptor and a quicker dissociation rate than ET1. ET3 was unable to desensitize endothelin A receptor and the neurokinin A receptor; this is in contrast to ET1 that desensitizes both receptors. These results suggests that the receptors that undergo homologous desensitization are able to heterologously desensitize other receptors that activate PLC-beta. Furthermore, the agonist-specific dissociation constant dictates the extent of desensitization and time of recovery of the receptor-mediated response
— id: 12660, year: 1996, vol: 6, page: 99, stat: Journal Article,

Developmental expression and functional characterization of the potassium-channel subunit Kv3.1b in parvalbumin-containing interneurons of the rat hippocampus
Du J; Zhang L; Weiser M; Rudy B; McBain CJ
1996 Jan 15;16(2):506-518, Journal of neuroscience
The expression of the voltage-gated K(+)-channel subunit Kv3.1b in the developing hippocampus was determined by immunoblot and immunohistochemical techniques. Kv3.1b protein was detected first at postnatal day (P) 8. The Kv3.1b-immunopositive cell number per tissue section reached a maximum at P14 and was maintained through P40. In contrast, the Kv3.1b protein content of isolated membrane vesicles in immunoblots progressively increased through P40, suggesting an increase in Kv3.1b content per cell throughout this time period. Kv3.1b protein was expressed selectively in the somata, proximal dendrites, and axons of cells lying within or near the pyramidal cell layer, consistent with their being GABAergic inhibitory interneurons. Kv3.1b was present in approximately 80% of parvalbumin-positive interneurons. The developmental onset of Kv3.1b and parvalbumin immunoreactivity was identical. In contrast, Kv3.1b was mostly absent from the subset of somatostatin-positive inhibitory interneurons. Electrophysiological recordings were made from stratum pyramidale interneurons in which morphology and Kv3.1b-positive immunoreactivity were confirmed post hoc. Outward currents had voltage-dependent and biophysical properties resembling those of channels formed by Kv3.1b. The current blocked by low concentrations of 4-aminopyridine (4-AP) showed marked inactivation, suggesting that Kv3.1b may coassemble with other members of the Kv3 subfamily. In current-clamp recordings, concentrations of 4-AP that blocked the current through Kv3.1b channels allowed us tentatively to assign a role to Kv3.1b-containing channels in action-potential repolarization. These data demonstrate that Kv3.1b is regulated developmentally in a specific subpopulation of hippocampal interneurons and that channels containing this subunit may be a major determinant in imparting 'fast-spiking' characteristics to these and other cells throughout the central nervous system containing the Kv3.1b subunit
— id: 18832, year: 1996, vol: 16, page: 506, stat: Journal Article,

Identification of voltage-gated K+ channels containing Kv3 subunits in neurons from the globus pallidus
Hernandez-Pineda, R.; Hernandez-Cruz, A.; Moreno, H.; Chow, A.; Rudy, B.
1996 ;22(1-3):1754-1754, Abstracts (Society for Neuroscience)
— id: 92548, year: 1996, vol: 22, page: 1754, stat: Journal Article,

G-protein-gated inward rectifier K+ channel proteins (GIRK1) are present in the soma and dendrites as well as in nerve terminals of specific neurons in the brain
Ponce A; Bueno E; Kentros C; Vega-Saenz de Miera E; Chow A; Hillman D; Chen S; Zhu L; Wu MB; Wu X; Rudy B; Thornhill WB
1996 Mar 15;16(6):1990-2001, Journal of neuroscience
G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors
— id: 7033, year: 1996, vol: 16, page: 1990, stat: Journal Article,

K+ channel subunit isoforms with divergent carboxy-terminal sequences carry distinct membrane targeting signals
Ponce, A; Rudy, B; deMiera, VS
1996 DEC ;7(3):1466-1466, Molecular biology of the cell
— id: 53354, year: 1996, vol: 7, page: 1466, stat: Journal Article,

Cloning of a novel component of A-type K+ channels operating at subthreshold potentials with unique expression in heart and brain
Serodio P; Vega-Saenz de Miera E; Rudy B
1996 May;75(5):2174-2179, Journal of neurophysiology
1. Proteins of the Kv4 or Shal-related subfamily are key components of transient K+ channels (A channels) operating at subthreshold values of the membrane potential. We have cloned and characterized a new mammalian Kv4 or Shal-related cDNA (Kv4.3) that predicts a protein with strong sequence conservation with the other known members of this subfamily. 2. Injection of Kv4.3 transcripts into Xenopus oocytes generates an A type K+ current, with small but physiologically significant differences from the currents expressed by Kv4.2 and Kv4.1 mRNAs. Kv4.3 currents can be modified to resemble native A currents by coinjection with a low molecular weight mRNA fraction from rat brain which does not express detectable currents on its own. Particularly striking is a 7-to-10-fold increase in the rate of recovery from inactivation, a 5- to 10-fold increase in current magnitude and a 3- to 4-fold increase in sensitivity to 4-amino pyridine (4-AP). 3. In situ hybridization histochemistry was used to compare the expression of the three known Kv4 genes. Kv4.2 and Kv4.3 (but not Kv4.1) are abundant in the adult rat brain, with each displaying a specific, but sometimes overlapping pattern of expression. Moreover, a reciprocal gradient of expression of Kv4.2 and Kv4.3 transcripts is seen in some brain areas, such as in the pyramidal cell layers of the hippocampus and the granule cell layer of the cerebellum. Therefore Kv4 proteins may form heteromultimeric channels of distinct subunit composition in different neurons. Moreover, the results suggest that neurons such as pyramidal cells in the hippocampus and granule cells in the cerebellum represent heterogeneous cell populations in terms of their ISA, and hence in their firing patterns. Kv4.2 and Kv4.3 also display complementary expression in the heart, with Kv4.3 being more abundant in atria and Kv4.2 in ventricle. The existence of multiple Kv4 proteins forming channels of variable subunit combinations helps explain the diversity of ISA channels in neurons
— id: 12610, year: 1996, vol: 75, page: 2174, stat: Journal Article,

Expression of K+ channel proteins in the medial nucleus of the trapezoid body
Strout, J.; Chow, A.; Thornhill, W.; Ellisman, M.; Rudy, B.
1996 ;22(1-3):1752-1752, Abstracts (Society for Neuroscience)
— id: 92549, year: 1996, vol: 22, page: 1752, stat: Journal Article,

NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines
Wang D; Youngson C; Wong V; Yeger H; Dinauer MC; Vega-Saenz Miera E; Rudy B; Cutz E
1996 Nov 12;93(23):13182-13187, Proceedings of the National Academy of Sciences of the United States of America
Pulmonary neuroepithelial bodies (NEB) are widely distributed throughout the airway mucosa of human and animal lungs. Based on the observation that NEB cells have a candidate oxygen sensor enzyme complex (NADPH oxidase) and an oxygen-sensitive K+ current, it has been suggested that NEB may function as airway chemoreceptors. Here we report that mRNAs for both the hydrogen peroxide sensitive voltage gated potassium channel subunit (KH2O2) KV3.3a and membrane components of NADPH oxidase (gp91phox and p22phox) are coexpressed in the NEB cells of fetal rabbit and neonatal human lungs. Using a microfluorometry and dihydrorhodamine 123 as a probe to assess H2O2 generation, NEB cells exhibited oxidase activity under basal conditions. The oxidase in NEB cells was significantly stimulated by exposure to phorbol esther (0.1 microM) and inhibited by diphenyliodonium (5 microM). Studies using whole-cell voltage clamp showed that the K+ current of cultured fetal rabbit NEB cells exhibited inactivating properties similar to KV3.3a transcripts expressed in Xenopus oocyte model. Exposure of NEB cells to hydrogen peroxide (H2O2, the dismuted by-product of the oxidase) under normoxia resulted in an increase of the outward K+ current indicating that H2O2 could be the transmitter modulating the O2-sensitive K+ channel. Expressed mRNAs or corresponding protein products for the NADPH oxidase membrane cytochrome b as well as mRNA encoding KV3.3a were identified in small cell lung carcinoma cell lines. The studies presented here provide strong evidence for an oxidase-O2 sensitive potassium channel molecular complex operating as an O2 sensor in NEB cells, which function as chemoreceptors in airways and in NEB related tumors. Such a complex may represent an evolutionary conserved biochemical link for a membrane bound O2-signaling mechanism proposed for other cells and life forms
— id: 18831, year: 1996, vol: 93, page: 13182, stat: Journal Article,

Developmental expression of KV3.2, KV3.1 and GIRK K+ channel proteins in the mammalian CNS
Bueno, E.; Yang, H.; Ponce, A.; Lau, D. H. P.; Chow, A.; Chen, S.; Rameau, G.; Sekirnjak, C.; Martone, M. E.; Ellisman, M.; Hillman, D.; Rudy, B.; Thornhill, W.
1995 ;21(1-3):1329-1329, Abstracts (Society for Neuroscience)
— id: 92259, year: 1995, vol: 21, page: 1329, stat: Journal Article,

Conserved cysteine residues in the cytoplasmic trail of the human neurokinin A receptor are involved in receptor desensitization
Cyr, C. R.; Josiah, S.; Rudy, B.; Devi, L.; Kris, R. M.
1995 ;21(1-3):1595-1595, Abstracts (Society for Neuroscience)
— id: 92551, year: 1995, vol: 21, page: 1595, stat: Journal Article,

MODULATION OF KV3.3 A TRANSIENT, HIGH VOLTAGE-ACTIVATING POTASSIUM CHANNEL, BY NITRIC-OXIDE
DEMIERA, EVS; RUDY, B
1995 NOV ;6(23):308-308, Molecular biology of the cell
— id: 52663, year: 1995, vol: 6, page: 308, stat: Journal Article,

Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions [see comments]
Lev S; Moreno H; Martinez R; Canoll P; Peles E; Musacchio JM; Plowman GD; Rudy B; Schlessinger J
1995 Aug 31;376(6543):737-745, Nature
The protein tyrosine kinase PYK2, which is highly expressed in the central nervous system, is rapidly phosphorylated on tyrosine residues in response to various stimuli that elevate the intracellular calcium concentration, as well as by protein kinase C activation. Activation of PYK2 leads to modulation of ion channel function and activation of the MAP kinase signalling pathway. PYK2 activation may provide a mechanism for a variety of short- and long-term calcium-dependent signalling events in the nervous system
— id: 7926, year: 1995, vol: 376, page: 737, stat: Journal Article,

Alternative splicing of the human Shaker K+ channel beta 1 gene and functional expression of the beta 2 gene product
McCormack K; McCormack T; Tanouye M; Rudy B; Stuhmer W
1995 Aug 14;370(1-2):32-36, FEBS letters
Mammalian voltage-activated Shaker K+ channels associate with at least three cytoplasmic proteins: Kv beta 1, Kv beta 2 and Kv beta 3. These beta subunits contain variable N-termini, which can modulate the inactivation of Shaker alpha subunits, but are homologous throughout an aldo-keto reductase core. Human and ferret beta 3 proteins are identical with rat beta 1 throughout the core while beta 2 proteins are not; beta 2 also contains a shorter N-terminus and has no reported physiological role. We report that human beta 1 and beta 3 are derived from the same gene and that beta 2 modulates the inactivation properties of Kv1.4 alpha subunits
— id: 18833, year: 1995, vol: 370, page: 32, stat: Journal Article,

Are K+ channel beta-subunit NAD(P)H-dependent oxidoreductase proteins?
McCormack, T.; McCormack, K.; Moreno, H.; Rudy, B.
1995 ;21(1-3):1827-1827, Abstracts (Society for Neuroscience)
— id: 92550, year: 1995, vol: 21, page: 1827, stat: Journal Article,

Thalamocortical projections have a K+ channel that is phosphorylated and modulated by cAMP-dependent protein kinase
Moreno H; Kentros C; Bueno E; Weiser M; Hernandez A; Vega-Saenz de Miera E; Ponce A; Thornhill W; Rudy B
1995 Aug;15(8):5486-5501, Journal of neuroscience
The finding that some K+ channel mRNAs are restricted to certain populations of neurons in the CNS suggests that there are K+ channels tailored to certain neuronal circuits. One such example are the transcripts from the KV3.2 gene, the majority of which are expressed in thalamic relay neurons. To gain insights into the specific roles of KV3.2 subunits, site specific antibodies were raised to determine their localization in thalamic relay neurons. Immunohistochemical and focal lesioning studies demonstrate that KV3.2 proteins are localized to the terminal fields of thalamocortical projections. It is also shown that KV3.2 channels expressed in vitro are strongly inhibited through phosphorylation by cAMP-dependent protein kinase (PKA). Channels containing KV3.1 subunits, which otherwise exhibit nearly identical electrophysiological properties in heterologous expression systems but have a different and less restricted pattern of expression in the CNS, are not affected by PKA. Therefore, this modulation might be associated with the specific roles of KV3.2 subunits. Furthermore, we demonstrate that KV3.2 proteins can be phosphorylated in situ by intrinsic PKA. KV3.2 subunits display properties and have a localization consistent with a role in the regulation of the efficacy of the thalamocortical synapse, and could thereby participate in the neurotransmitter-mediated control of functional states of the thalamocortical system associated with global states of awareness
— id: 6846, year: 1995, vol: 15, page: 5486, stat: Journal Article,

Nitric oxide and cGMP modulate a presynaptic K+ channel in vitro
Moreno, H.; Bueno, E.; Hernandez Cruz, A.; Ponce, A.; Rudy, B.
1995 ;21(1-3):506-506, Abstracts (Society for Neuroscience)
— id: 92553, year: 1995, vol: 21, page: 506, stat: Journal Article,

Alternatively Spliced Carboxyl-Termini Determine The Targeting Of KV3.2 Channels In MDCK Cells
Ponce, A.; Vega-Saenz De Miera, E.; Moreno, H.; Bueno, E.; Aleman, V.; Rudy, B.
1995 ;21(1-3):283-283, Abstracts (Society for Neuroscience)
— id: 92555, year: 1995, vol: 21, page: 283, stat: Journal Article,

Cloning, expression and distribution of KV4.3, a new mammalian subunit of A-type, low-voltage-activating potassium channels
Serodio, P.; Vega-Saenz De Miera, E.; Rudy, B.
1995 ;21(1-3):1328-1328, Abstracts (Society for Neuroscience)
— id: 92552, year: 1995, vol: 21, page: 1328, stat: Journal Article,

Phosphorylation may be required to activate Shaw related K+ channels
Vega-Saenz De Miera, E.; Moreno, H.; Rudy, B.
1995 ;21(1-3):505-505, Abstracts (Society for Neuroscience)
— id: 92554, year: 1995, vol: 21, page: 505, stat: Journal Article,

The potassium channel subunit KV3.1b is localized to somatic and axonal membranes of specific populations of CNS neurons
Weiser M; Bueno E; Sekirnjak C; Martone ME; Baker H; Hillman D; Chen S; Thornhill W; Ellisman M; Rudy B
1995 Jun;15(6):4298-4314, Journal of neuroscience
Potassium channels play major roles in the regulation of many aspects of neuronal excitability. These channels are particularly well suited for such multiplicity of roles since there is a large diversity of channel types. This diversity contributes to the ability of specific neurons (and possibly different regions of the same neuron) to respond uniquely to a given input. Neuronal integration depends on the local response of spatially segregated inputs to the cell and the communication of these integration centers with the axon. Therefore, the functional implications of a given set of K+ channels varies depending on their precise location on the neuronal surface. Site-specific antibodies were utilized to characterize the distribution of KV3.1b, a subunit of voltage-gated K+ channels in CNS neurons. KV3.1b subunits are expressed in specific neuronal populations of the rat brain, such as cerebellar granule cells, projecting neurons of deep cerebellar nuclei, the substantia nigra pars-reticulata, the globus pallidus, and the ventral thalamus (reticular thalamic nucleus, ventral lateral geniculate and zona incerta). The KV3.1b protein is also present in various neuronal populations involved in the processing of auditory signals, including the inferior colliculus, the nuclei of the lateral lemniscus, the superior olive, and some parts of the cochlear nuclei; as well as in several other neuronal groups in the brainstem (e.g., in the oculomotor nucleus, the pontine nuclei, the reticulotegmental nucleus of the pons, trigeminal and vestibular nuclei, and the reticular formation) and subsets of neurons in the neocortex, the hippocampus and the caudate-putamen shown by double staining to correspond to neurons containing parvalbumin. KV3.1b subunits are localized predominantly in somatic and axonal membranes (particularly in axonal terminal fields) but are much less prominent in dendritic arborizations. This distribution is different than that of other subunits of voltage gated K+ channels and is consistent with a role in the modulation of action potentials. KV3.1b proteins have a cellular and subcellular distribution different than the related KV3.2 subunits which express in Xenopus oocytes currents similar to those expressed by KV3.1b
— id: 6775, year: 1995, vol: 15, page: 4298, stat: Journal Article,

Heterologous desensitization of the human endothelin A and neurokinin a receptors in Xenopus laevis oocytes
Cyr, Curt R.; Rudy, Bernardo; Kris, Richard M.
1994 ;20(1-2):27-27, Abstracts (Society for Neuroscience)
— id: 92560, year: 1994, vol: 20, page: 27, stat: Journal Article,

Identification of a new Shab K+ channel mRNA in PC12 cells and rat atria
Lau, D.; Vega-Saenz De Miera, E.; Sen, K.; Rudy, B.; Wu, M. Bin; Thornhill, W.
1994 ;20(1-2):75-75, Abstracts (Society for Neuroscience)
— id: 92559, year: 1994, vol: 20, page: 75, stat: Journal Article,

Protein kinase A modulates a voltage gated K+ channel protein present in thalamo-cortical projections
Moreno, H.; Hernandez-Cruz, A.; Bueno, E.; Vega-Saenz De Miera, E.; Kentros, C.; Podberesky, D.; Rudy, B.
1994 ;20(1-2):725-725, Abstracts (Society for Neuroscience)
— id: 92557, year: 1994, vol: 20, page: 725, stat: Journal Article,

Molecular characterization of the sodium channels expressed in mammalian cerebellar Purkinje cells
Rudy, B.; Vega-Saenz De Miera, E.; Sugimori, M.; Llinas, R.
1994 ;20(1-2):64-64, Abstracts (Society for Neuroscience)
— id: 92350, year: 1994, vol: 20, page: 64, stat: Journal Article,

Identification of molecular components of A-type channels activating at subthreshold potentials
Serodio P; Kentros C; Rudy B
1994 Oct;72(4):1516-1529, Journal of neurophysiology
1. Xenopus oocytes injected with rat brain mRNA express a transient K+ current similar to the A current that activates transiently near the threshold for Na+ action potential generation (ISA) seen in somatic recordings from neurons. We used hybrid arrest with antisense oligonucleotides to investigate which of the cloned K+ channel proteins might be components of the channels responsible for the ISA expressed from brain mRNA. An oligonucleotide complementary to a sequence common to all known mammalian Shal-related mRNAs [KV4.1, KV4.2, and KV4.3 (the nomenclature of Sh K+ channel genes of Chandy and colleagues was used in this paper)] blocked the expression of the ISA. An oligonucleotide complementary only to the KV4.2 mRNA, the most abundant Shal-related transcript in rat brain RNA preparations, was also quite efficient in arresting the expression of the ISA from brain. These experiments indicate that Shal-related proteins are important components of the channels carrying the ISA expressed in oocytes injected with brain mRNA. However, there are several significant differences between this ISA and the currents expressed in the same oocytes by in vitro transcribed KV4.1 or KV4.2 cRNA. Most of these differences are eliminated if KV4.1 or KV4.2 cRNA is coinjected with brain poly-(A) RNA treated with antisense oligonucleotides which arrest the expression of the ISA, or with a 2-4Kb rat brain poly-(A) RNA fraction which does not express detectable K+ currents under the same recording conditions. These data support the hypothesis that ISA channels such as those expressed from brain mRNA contain Shal proteins that can be modified by proteins encoded in RNAs that by themselves do not express K+ currents
— id: 6748, year: 1994, vol: 72, page: 1516, stat: Journal Article,

Identification of molecular components of A-type K+ channels activating at subthreshold potentials
Serodio, P.; Rudy, B.
1994 ;20(1-2):77-77, Abstracts (Society for Neuroscience)
— id: 92558, year: 1994, vol: 20, page: 77, stat: Journal Article,

Clustering of six human 11p15 gene homologs within a 500-kb interval of proximal mouse chromosome 7
Stubbs L; Rinchik EM; Goldberg E; Rudy B; Handel MA; Johnson D
1994 Nov 15;24(2):324-332, Genomics
Homologs of genes mapping to human chromosome 11p15 are located in three distinct, widely separated regions of mouse chromosome 7 (Mmu7). To date, six genes have been localized to the most proximal HSA11p15/Mmu7 homology region, including Ldh3 (encoding lactate dehydrogenase C), Ldh1 (lactate dehydrogenase A), Myod1 (myogenic differentiation factor-1), Tph (tryptophan hydroxylase), Saa1 (serum amyloid-A-1), and Kcnc1 (encoding a Shaw-type voltage-gated potassium channel). To define the overall size and organization of this region of Mmu7, we have established a long-range physical map including the murine Ldh1, Ldh3, Saa, Tph, Kcnc1, and Myod1 genes. Our results demonstrate that these six genes are physically clustered and are distributed throughout a 500-kb interval located just proximal of the pink-eyed dilution (p) locus. These data, together with recent mapping studies within the related region of HSA11p15, demonstrate that gene content and organization within this proximal homology segment have been highly conserved throughout evolution
— id: 18834, year: 1994, vol: 24, page: 324, stat: Journal Article,

Modulation of KV3.3 K+ channels by oxidation and phosphorylation
Vega-Saenz De Miera, E.; Moreno, H.; Rudy, B.
1994 ;20(1-2):725-725, Abstracts (Society for Neuroscience)
— id: 92556, year: 1994, vol: 20, page: 725, stat: Journal Article,

Differential expression of Shaw-related K+ channels in the rat central nervous system
Weiser M; Vega-Saenz de Miera E; Kentros C; Moreno H; Franzen L; Hillman D; Baker H; Rudy B
1994 Mar;14(3 Pt 1):949-972, Journal of neuroscience
The family of mammalian genes related to the Drosophila Shaker gene, consisting of four subfamilies, is thought to encode subunits of tetrameric voltage-gated K+ channels. There is compelling evidence that subunits of the same subfamily, but not of different subfamilies, form heteromultimeric channels in vitro, and thus, each gene subfamily is postulated to encode components of an independent channel system. In order to identify cells with native channels containing subunits of one of these subfamilies (Shaw-related or ShIII), the cellular distribution of ShIII transcripts was examined by Northern blot analysis and in situ hybridization. Three of four ShIII genes (KV3.1, KV3.2, and KV3.3) are expressed mainly in the CNS. KV3.4 transcripts are also present in the CNS but are more abundant in skeletal muscle. In situ hybridization studies in the CNS reveal discrete and specific neuronal populations that prominently express ShIII mRNAs, both in projecting and in local circuit neurons. In the cerebral cortex, hippocampus, and caudate-putamen, subsets of neurons can be distinguished by the expression of specific ShIII mRNAs. Each ShIII gene exhibits a unique pattern of expression; however, many neuronal populations expressing KV3.1 transcripts also express KV3.3 mRNAs. Furthermore, KV3.4 transcripts are present, albeit at lower levels, in several of the neuronal populations that also express KV3.1 and/or KV3.3 mRNAs, revealing a high potential for heteromultimer formation between the products of three of the four genes. Expression of ShIII cRNAs in Xenopus oocytes was used to explore the functional consequences of heteromultimer formation between ShIII subunits. Small amounts of KV3.4 cRNA, which expresses small, fast-inactivating currents when injected alone, produced fast-inactivating currents that are severalfold larger when coinjected with an excess of KV3.1 or KV3.3 cRNA. This amplification is due to both an increase in single-channel conductance in the heteromultimeric channels and the observation that less than four, perhaps even a single KV3.4 subunit is sufficient to impart fast-inactivating properties to the channel. The oocyte experiments indicate that the apparently limited, low-level expression of KV3.4 in the CNS is potentially significant. The anatomical studies suggest that heteromultimer formation between ShIII proteins might be a common feature in the CNS. Moreover, the possibility that the subunit composition of heteromultimers varies in different neurons should be considered, since the ratios of overlapping signals change from one neuronal population to another. In order to proceed with functional analysis of native ShIII channels, it is important to known which subunit compositions might occur in vivo. The studies presented here provide important clues for the identification of native homo- and heteromultimeric ShIII channels in neurons
— id: 6552, year: 1994, vol: 14, page: 949, stat: Journal Article,

Immunocytochemical evidence for co-localization of an alternatively spliced shaw related K+ channel
Weiser, M.; Bueno, E.; Hillman, D.; Yang, T.; Baker, H.; Ellisman, M.; Thornhill, B.; Rudy, B.
1994 ;20(1-2):863-863, Abstracts (Society for Neuroscience)
— id: 92260, year: 1994, vol: 20, page: 863, stat: Journal Article,

Prolonged desensitization of the human endothelin A receptor in Xenopus oocytes. Comparative studies with the human neurokinin A receptor
Cyr CR; Rudy B; Kris RM
1993 Dec 15;268(35):26071-26074, Journal of biological chemistry
Human endothelin (ET) A receptor (hETAR) is a G-protein-mediated receptor that binds ET1 with high affinity and ET2 and ET3 with lower affinities. ET1 is the most potent endogenous vasoconstrictor known at this time. When expressed in Xenopus laevis oocytes, hETAR is rapidly desensitized after stimulation with ET1. This desensitization lasts 90-110 min. Human neurokinin A (hNKAR) and human serotonin type 2 receptors were also expressed in the Xenopus system for comparison to hETAR. hNKAR desensitizes for 25-35 min, while the serotonin receptor does not appear to desensitize. To examine the role of the cytoplasmic tail of hETAR in desensitization, deletion mutations were constructed which remove 11, 36, and 51 amino acids from the cytoplasmic tail. The mutations removing 11 and 36 residues were functional, but the mutation removing 51 amino acids was not functional. The two functional mutations have a resensitization time similar to that of hETAR. In summary, the prolonged desensitization time of hETAR is unique for G-protein-mediated receptors and may attenuate the adverse physiological effects of the endothelin family. In addition, the cytoplasmic tail of hETAR does not appear to play a role in desensitization or resensitization of this receptor
— id: 6349, year: 1993, vol: 268, page: 26071, stat: Journal Article,

Localization of Shaw-related K+ channel genes on mouse and human chromosomes
Haas M; Ward DC; Lee J; Roses AD; Clarke V; D'Eustachio P; Lau D; Vega-Saenz de Miera E; Rudy B
1993 Dec;4(12):711-715, Mammalian genome
Four related genes, Shaker, Shab, Shaw, and Shal, encode voltage-gated K+ channels in Drosophila. Multigene subfamilies corresponding to each of these Drosophila genes have been identified in rodents and primates; this suggests that the four genes are older than the common ancestor of present-day insects and mammals and that the expansion of each into a family occurred before the divergence of rodents and primates. In order to define these evolutionary relationships more precisely and to facilitate the search for mammalian candidate K+ channel gene mutations, we have mapped members of the Shaw-homologous gene family in humans and mice. Fluorescence in situ hybridization analysis of human metaphase chromosomes mapped KCNC2 (KShIIIA, KV3.2) and KCNC3 (KShIIID, KV3.3) to Chromosome (Chr) 19q13.3-q13.4. Inheritance patterns of DNA restriction fragment length variants in recombinant inbred strains of mice placed the homologous mouse genes on distal Chr 10 near Ms15-8 and Mdm-1. The mouse Kcnc1 (KShIIIB, NGK2-KV4, KV3.1) gene mapped to Chr7 near Tam-1. These results are consistent with the hypothesis that the generation of the mammalian KCNC gene family included both duplication events to generate family members in tandem arrays (KCNC2, KCNC3) and dispersion of family members to unlinked chromosomal sites (KCNC1). The KNCN2 and KCNC3 genes define a new synteny group between humans and mice
— id: 17235, year: 1993, vol: 4, page: 711, stat: Journal Article,

Localization of a highly conserved human potassium channel gene (NGK2-KV4; KCNC1) to chromosome 11p15
Ried T; Rudy B; Vega-Saenz de Miera E; Lau D; Ward DC; Sen K
1993 Feb;15(2):405-411, Genomics
Several genes (the Shaker or Sh gene family) encoding components of voltage-gated K+ channels have been identified in various species. Based on sequence similarities Sh genes are classified into four groups or subfamilies. Mammalian genes of each one of these subfamilies also show high levels of sequence similarity to one of four related Drosophila genes: Shaker, Shab, Shaw, and Shal. Here we report the isolation of human cDNAs for a Shaw-related product (NGK2, KV3.1a) previously identified in rat and mice. A comparison of the nucleotide and deduced amino acid sequence of NGK2 in rodents and humans shows that this product is highly conserved in mammals; the human NGK2 protein shows over 99% amino acid sequence identity to its rodent homologue. The gene (NGK2-KV4; KCNC1) encoding NGK2 was mapped to human chromosome 11p15 by fluorescence in situ hybridization with the human NGK2 cDNAs
— id: 18835, year: 1993, vol: 15, page: 405, stat: Journal Article,

Analysis of the spatial distribution and co-expression of Shaw related K+ channels in the rat central nervous system
Weiser, M.; Vega-Saenz De Miera, E.; Kentros, C.; Moreno, H.; Baker, H.; Rudy, B.
1993 ;19(1-3):1063-1063, Abstracts (Society for Neuroscience)
— id: 92561, year: 1993, vol: 19, page: 1063, stat: Journal Article,

Analysis of products of two genes encoding high-voltage activating, tea-sensitive, A-type potassium currents
De Miera, E. Vega-Saenz; Moreno, H.; Sen, K.; Lau, D.; Rudy, B.
1992 ;18(1-2):77-77, Abstracts (Society for Neuroscience)
— id: 92565, year: 1992, vol: 18, page: 77, stat: Journal Article,

MODULATION OF CLONED VOLTAGE-GATED K+ CHANNELS BY HYDROGEN-PEROXIDE
DEMEIRA, EVS; RUDY, B
1992 SEP ;3(4):A301-A301, Molecular biology of the cell
— id: 51867, year: 1992, vol: 3, page: A301, stat: Journal Article,

Alternative splicing of the 5'-untranslated region of a gene encoding potassium channel components
Kentros, C.; Weiser, M.; De Miera, E. Vega-Saenz; Morel, K.; Baker, H.; Rudy, B.
1992 ;18(1-2):1093-1093, Abstracts (Society for Neuroscience)
— id: 92562, year: 1992, vol: 18, page: 1093, stat: Journal Article,

Region-specific expression of a K+ channel gene in brain
Rudy B; Kentros C; Weiser M; Fruhling D; Serodio P; Vega-Saenz de Miera E; Ellisman MH; Pollock JA; Baker H
1992 May 15;89(10):4603-4607, Proceedings of the National Academy of Sciences of the United States of America
Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions
— id: 13595, year: 1992, vol: 89, page: 4603, stat: Journal Article,

DRK1 mRNA is induced by NGF in rat pheochromocytoma PC12 cells
Rudy, B.; Lau, D.; Lin, J. W.; Pollack, J.; Kentros, C.
1992 ;18(1-2):77-77, Abstracts (Society for Neuroscience)
— id: 92564, year: 1992, vol: 18, page: 77, stat: Journal Article,

Ion channels
Rudy, Bernardo; Iverson, Linda E
San Diego : Academic Press, c1992,
— id: 403, year: 1992, vol: , page: , stat: ,

Molecular components of low-voltage-activating A currents
Serodio, P.; De Miera, E. Vega-Saenz; Lau, D.; Rudy, B.
1992 ;18(1-2):78-78, Abstracts (Society for Neuroscience)
— id: 92563, year: 1992, vol: 18, page: 78, stat: Journal Article,

Cloning of ShIII (Shaw-like) cDNAs encoding a novel high-voltage-activating, TEA-sensitive, type-A K+ channel
Vega-Saenz de Miera E; Moreno H; Fruhling D; Kentros C; Rudy B
1992 Apr 22;248(1321):9-18, Proceedings of the Royal Society. B. Biological sciences
Transient voltage-dependent potassium (K+) currents, also known as A currents, have been of great interest to neurophysiologists due to their special roles in neuronal excitability. Several cDNAs encoding transcripts expressing A currents have been characterized. Recently, a cDNA (KShIIIC or Raw3) was isolated which expresses an unusual A current that is highly sensitive to TEA, and activates at potentials more positive than -20 mV. Channels containing this protein may have specialized roles in modulating the electrical behaviour of neurons. Here we report the isolation and characterization of two rat cDNAs corresponding to two alternatively spliced transcripts (KShIIID.1 and KShIIID.2) from another gene (KShIIID) of the same subfamily as KShIIIC, the ShIII or Shaw-related gene subfamily. KShIIID.1 also expresses an unusual high-voltage-activating, TEA-sensitive A-type channel. There are, however, significant differences between KShIIIC and KShIIID channels which may have interesting functional consequences. The two most important differences are: (i) KShIIID channels conduct in the steady-state over a much broader window of potentials than KShIIIC; this reflects differences between the kinetic schemes of the two channels; and (ii) KShIIID inactivates with significantly slower kinetics than KShIIIC. The identification of KShIIID transcripts contributes to our knowledge of the molecular components that may determine the functional diversity of A currents and provides exciting opportunities to increase our understanding of the structure and function of K+ channels
— id: 13627, year: 1992, vol: 248, page: 9, stat: Journal Article,

Modulation of K+ channels by hydrogen peroxide
Vega-Saenz de Miera E; Rudy B
1992 Aug 14;186(3):1681-1687, Biochemical & biophysical research communications
External application of hydrogen peroxide (H2O2) was found to inhibit the time-dependent fast inactivation process of three cloned voltage-gated K+ channels expressed in Xenopus oocytes: KShIIIC, KShIIID and HukII. As expected from kinetic models where some channels are still opening while a significant fraction of channels is already inactivated there was a large increase in current magnitude concomitant to inactivation block. The channels otherwise functioned normally. The effects of H2O2 were specific (other cloned voltage-gated K+ channels were not affected), and reversible, the currents returned to normal upon removal of the H2O2. H2O2 is produced during normal metabolism; it could act as a modulator of excitability through effects on K+ channels if effective local concentrations are reached in neuronal regions close to the channel. KShIIIC and KShIIID currents are very similar to an O2-sensitive K+ current present in type I cells of the carotid body which is believed to underlie the modulation of excitability of these cells by changes in arterial O2 pressure. H2O2 has been proposed as an intermediary between O2 and cellular response in the carotid body; our results provide support for this model
— id: 13479, year: 1992, vol: 186, page: 1681, stat: Journal Article,

Characterization of maintained voltage-dependent K(+)-channels induced in Xenopus oocytes by rat brain mRNA
Hoger JH; Rudy B; Lester HA; Davidson N
1991 Apr;10(1):1-11, Brain research. Molecular brain research
The voltage-dependent K+ currents encoded by rat brain mRNA were studied in Xenopus oocytes after the voltage-dependent Na+ currents and the Ca(2+)-activated Cl- currents were eliminated pharmacologically. This paper describes the maintained K+ currents (IK), defined primarily by resistance to inactivation for 1 s at a holding potential of -40 mV. IK activates at potentials more positive than -60 to -70 mV and consists of both low-threshold and high-threshold components. IK is partially blocked by both tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which appear to be blocking the same component. Long depolarizing pulses result in incomplete inactivation of IK; the inactivating component is inhibited by TEA. Sucrose density gradient fractionation partially resolves the RNA encoding the several components of IK; most IK arises from size classes between 3.8 and 9.5 kb. The study gives further evidence for the existence of numerous distinct RNA populations that encode brain K+ channels different from previously reported cloned K+ channels that have been expressed in Xenopus oocytes
— id: 18838, year: 1991, vol: 10, page: 1, stat: Journal Article,

A role for hydrophobic residues in the voltage-dependent gating of Shaker K+ channels
McCormack K; Tanouye MA; Iverson LE; Lin JW; Ramaswami M; McCormack T; Campanelli JT; Mathew MK; Rudy B
1991 Apr 1;88(7):2931-2935, Proceedings of the National Academy of Sciences of the United States of America
A leucine heptad repeat is well conserved in voltage-dependent ion channels. Herein we examine the role of the repeat region in Shaker K+ channels through substitution of the leucines in the repeat and through coexpression of normal and truncated products. In contrast to leucine-zipper DNA-binding proteins, we find that the subunit assembly of Shaker does not depend on the leucine heptad repeat. Instead, we report that substitutions of the leucines in the repeat produce large effects on the observed voltage dependence of conductance voltage and prepulse inactivation curves. Our results suggest that the leucines mediate interactions that play an important role in the transduction of charge movement into channel opening and closing
— id: 18837, year: 1991, vol: 88, page: 2931, stat: Journal Article,

Molecular cloning of a member of a third class of Shaker-family K+ channel genes in mammals
McCormack T; Vega-Saenz de Miera EC; Rudy B
1991 May 1;88(9):4060-4060, Proceedings of the National Academy of Sciences of the United States of America
In the human proteolipid protein gene, the base sequence of the intronic region 5' to exon 6 was found to be 5'-ctctttcattttcctgcag-3' and not 5'-ctctttt-cattttcctgcag-3' as previously reported
— id: 18836, year: 1991, vol: 88, page: 4060, stat: Journal Article,

Cloning of a human cDNA expressing a high voltage-activating, TEA-sensitive, type-A K+ channel which maps to chromosome 1 band p21
Rudy B; Sen K; Vega-Saenz de Miera E; Lau D; Ried T; Ward DC
1991 Jul;29(3):401-412, Journal of neuroscience research
Over ten different mammalian genes related to the Drosophila Shaker gene (the Sh gene family) have been identified recently. These genes encode subunits of voltage-dependent K+ channels. The family consists of four subfamilies: ShI genes are homologues of Shaker; ShII, ShIII, and ShIV are homologues of three other Shaker-like genes in Drosophila, Shab, Shaw, and Shal, respectively. We report here the cloning of a human K+ channel ShIII cDNA (HKShIIIC) obtained from a brain stem cDNA library. HKShIIIC transcripts express an atypical voltage-dependent transient (A-type) K+ current in Xenopus oocytes. This current is activated by large membrane depolarizations and is extremely sensitive to the K+ channel blocker TEA unlike most A-type currents. The gene encoding HKShIIIC maps to chromosome 1p21
— id: 13974, year: 1991, vol: 29, page: 401, stat: Journal Article,

Families of potassium channel genes in mammals: Toward an understanding of the molecular basis of potassium channel diversity
Rudy, B; Kentros, C; Vela-Saenz De Miera, E
1991 Apr;2(2):89-102, Molecular & cellular neurosciences
— id: 105342, year: 1991, vol: 2, page: 89, stat: Journal Article,

The role of the divergent amino and carboxyl domains on the inactivation properties of potassium channels derived from the Shaker gene of Drosophila
Iverson LE; Rudy B
1990 Sep;10(9):2903-2916, Journal of neuroscience
Several products generated from the Drosophila Shaker gene by alternative splicing predict a group of similar proteins with an identical central and variable amino and carboxyl domains. We have constructed 9 Sh cDNAs combining 3 different 5' domains with 3 different 3' domains. RNA transcribed from 6 of these cDNAs induce K+ currents in Xenopus oocytes. All currents share similar properties of voltage dependence, potassium selectivity, and block by 4-AP, TEA, and charybdotoxin. These properties presumably result from a channel core formed by the identical central region of the proteins. The currents differ in macroscopic inactivation kinetics. Five RNAs induced K+ currents which inactivate, each at distinct rates, during short depolarizations. The sixth RNA induces a current that essentially does not inactivate unless depolarized for many seconds. This raises the possibility that Sh may encode nontransient as well as transient K+ currents. Analysis of currents produced by the various combinations suggests that the divergent amino domains influence the stability of a first, nonabsorbing, inactivated state. This results in striking differences in the probability of channel reopening, as observed in single-channel recordings, of those channels with identical carboxyl but different amino domains. Furthermore, based on macroscopic analysis of the currents, we suggest that the primary role of the carboxyl domains is to influence the relative stability between the first and a second inactivated state. The second inactivated state is essentially absorbing, and recovery from this state is very slow. The observed differences in the rates of recovery from inactivation of channels containing different carboxyl domains reflect differences in the rates at which they enter this second inactivated state
— id: 18840, year: 1990, vol: 10, page: 2903, stat: Journal Article,

Funnel-web spider venom and a toxin fraction block calcium current expressed from rat brain mRNA in Xenopus oocytes
Lin JW; Rudy B; Llinas R
1990 Jun;87(12):4538-4542, Proceedings of the National Academy of Sciences of the United States of America
Injection of rat brain mRNA into Xenopus oocytes has been shown to induce a calcium current (ICa) that is insensitive to dihydropyridine and omega-conotoxin. We examined the effect of funnel-web spider venom on two aspects of this expressed ICa: (i) the calcium-activated chloride current [ICl(Ca)] and (ii) the currents carried by barium ions through calcium channels (IBa). In the presence of 1.8 mM extracellular calcium, ICl(Ca) tail current became detectable between -30 and -40 mV from a holding potential of -80 mV and reached a maximal amplitude between 0 and +10 mV. Total spider venom partially (83%) and reversibly blocked the calcium-activated chloride current without changing its voltage sensitivity. A chromatographic toxin fraction from the venom also blocked this current (64%). The venom had a minimal effect on INa and IK. Direct investigation of inward current mediated by calcium channels was carried out in high-barium solution. IBa had a higher threshold of activation (-30 to -20 mV) and reached its maximal amplitude at about +20 mV. Total venom or a partly purified chromatographic toxic fraction blocked IBa partially and reversibly without changing its current-voltage characteristics. Furthermore, the extent of the total venom block depended on the concentration of extracellular barium. Only 35% of the IBa was blocked in 60 mM Ba2+, whereas the block increased to 65% and 71%, respectively, for 40 and 20 mM Ba2+. On the basis of these results, we propose that the calcium channels expressed from rat brain mRNA in Xenopus oocytes is similar to the recently discovered P-type channels
— id: 9922, year: 1990, vol: 87, page: 4538, stat: Journal Article,

MUTATIONS IN THE LEUCINE-HEPTAD REPEAT OF SHAKER POTASSIUM CHANNELS ALTER VOLTAGE-DEPENDENT GATING
MCCORMACK K; LIN J W; MCCORMACK T; MATHEW M K; TANOUYE M; RUDY B
1990 ;16(1):4-4, Abstracts (Society for Neuroscience)
— id: 92566, year: 1990, vol: 16, page: 4, stat: Journal Article,

Shaker K+ channel subunits from heteromultimeric channels with novel functional properties
McCormack K; Lin JW; Iverson LE; Rudy B
1990 Sep 28;171(3):1361-1371, Biochemical & biophysical research communications
A large number of related genes (the Sh gene family) encode potassium channel subunits which form voltage-dependent K+ channels by aggregating into homomulitimers. One of these genes, the Shaker gene in Drosophila, generates several products by alternative splicing. These products encode proteins with a constant central region flanked by variable amino and carboxyl domains. Coinjection of two Shaker RNAs with different amino or different carboxyl ends into Xenopus oocytes produces K+ currents that display functional properties distinct from those observed when each RNA is injected separately, indicating the formation of heteromultimeric channels. The analysis of Shaker heteromultimers suggests certain rules regarding the roles of variable amino and carboxyl domains in determining kinetic properties of heteromultimeric channels. Heteromultimers with different amino ends produce currents in which the amino end that produces more inactivation dominates the kinetics. In contrast, heteromultimers with different carboxyl ends recover from inactivation at a rate closer to that observed in homomultimers of the subunit which results in faster recovery. While this and other recent reports demonstrate that closely related Sh family proteins form functional heteromultimers, we show here that two less closely related Sh proteins do not seem to form functional heteromultimeric channels. The data suggest that sites for subunit recognition may be found in sequences within a core region, starting about 130 residues before the first membrane spanning domain of Shaker and ending after the last membrane spanning domain, which are not conserved between Sh Class I and Class III genes
— id: 18839, year: 1990, vol: 171, page: 1361, stat: Journal Article,

Molecular cloning of a member of a third class of Shaker-family K+ channel genes in mammals [published erratum appears in Proc Natl Acad Sci U S A 1991 May 1;88(9):4060]
McCormack T; Vega-Saenz de Miera EC; Rudy B
1990 Jul;87(13):5227-5231, Proceedings of the National Academy of Sciences of the United States of America
We report the cloning of RKShIIIA, a cDNA encoding a K+ channel sequence expressed in rat brain. This cDNA encodes K+ channel subunits that express in Xenopus oocytes a slow, 4-aminopyridine- and tetraethylammonium-sensitive, delayed rectifier-type K+ channel activated by large membrane depolarizations. This gene belongs to the Shaker (Sh) family of K+ channel genes, since the predicted protein has the same overall structure and shows significant homology to other members of this family. However, RKShIIIA cannot be assigned to either of the two known classes of Sh-family genes in mammals based on sequence analysis. Notable features of the RKShIIIA protein product include a probable cytoplasmic loop rich in prolines and a stretch very homologous to the Drosophila Shaw protein, both near the amino terminus
— id: 8472, year: 1990, vol: 87, page: 5227, stat: Journal Article,

HETEROMULTIMER FORMATION CAN PRODUCE A LARGE NUMBER OF DISTINCT K-CHANNELS
Mccormack, K; Lin, JW; Ramaswami, M; Tanouye, M; Iverson, L; Rudy, B
1990 Feb;57(2):A209-A209, Biophysical journal
— id: 32011, year: 1990, vol: 57, page: A209, stat: Journal Article,

MUTAGENESIS OF SHAKER POTASSIUM CHANNELS - WHATS BEHIND THE ZIPPER
Mccormack, K; Rudy, B; Ramaswami, M; Mathew, MK; Iverson, LE; Mccormack, TJ; Tanouye, M
1990 Feb;57(2):A210-A210, Biophysical journal
— id: 32012, year: 1990, vol: 57, page: A210, stat: Journal Article,

Differential effects of NGF, FGF, EGF, cAMP, and dexamethasone on neurite outgrowth and sodium channel expression in PC12 cells
Pollock JD; Krempin M; Rudy B
1990 Aug;10(8):2626-2637, Journal of neuroscience
PC12 cells are a pheochromocytoma cell line that can be made to differentiate into sympatheticlike neurons by nerve growth factor (NGF). An essential component of the NGF-induced differentiation is the development of action potentials and sodium channels. Using whole-cell clamp we have confirmed that NGF produces a 5- to 6-fold increase in sodium channel density. The sodium channels induced by NGF are not different from those in cells not treated with NGF and are similar to those in other cell types. Basic fibroblast growth factor (FGF), another growth factor that causes PC12 cells to differentiate into sympathetic-like neurons, also produces a 5- to 6-fold increase in sodium current density with channels indistinguishable from those in PC12 cells treated and not treated with NGF. Basic FGF produces the same or somewhat larger increase in sodium channel density but much less neurite outgrowth. In contrast, epidermal growth factor does not produce neurite outgrowth but induces a small, reproducible increase in sodium channel density. Cyclic AMP produces spike-like processes but not neurites and results in a decrease in sodium current and sodium current density. Dexamethasone, a synthetic glucocorticoid, inhibits the increase in sodium current and sodium current density but does not antagonize the neurite outgrowth induced by NGF. Thus, although the increase in sodium channel expression induced by NGF and basic FGF parallels the changes in morphology that lead to neurite outgrowth, it clearly does not depend on them. The results show that different aspects of neuronal differentiation might be independently regulated by the microenvironment
— id: 8471, year: 1990, vol: 10, page: 2626, stat: Journal Article,

DESCRIPTION OF A NEW CLASS OF POTASSIUM CHANNEL GENES
VEGA-SAENZ DE MIERA E; SEN K; SERODIO P; MCCORMACK T; RUDY B
1990 ;16(1):3-3, Abstracts (Society for Neuroscience)
— id: 92567, year: 1990, vol: 16, page: 3, stat: Journal Article,

SIZE FRACTIONATED RAT BRAIN MESSENGER RNA INDUCES VARIOUS VOLTAGE DEPENDENT POTASSIUM CHANNELS IN XENOPUS OOCYTES
HOGER J H; RUDY B; DAVIDSON N; LESTER H
1989 ;15(1):540-540, Abstracts (Society for Neuroscience)
— id: 92569, year: 1989, vol: 15, page: 540, stat: Journal Article,

Identification of genes from pattern formation, tyrosine kinase, and potassium channel families by DNA amplification
Kamb A; Weir M; Rudy B; Varmus H; Kenyon C
1989 Jun;86(12):4372-4376, Proceedings of the National Academy of Sciences of the United States of America
The study of gene family members has been aided by the isolation of related genes on the basis of DNA homology. We have adapted the polymerase chain reaction to screen animal genomes very rapidly and reliably for likely gene family members. Using conserved amino acid sequences to design degenerate oligonucleotide primers, we have shown that the genome of the nematode Caenorhabditis elegans contains sequences homologous to many Drosophila genes involved in pattern formation, including the segment polarity gene wingless (vertebrate int-1), and homeobox sequences characteristic of the Antennapedia, engrailed, and paired families. In addition, we have used this method to show that C. elegans contains at least five different sequences homologous to genes in the tyrosine kinase family. Lastly, we have isolated six potassium channel sequences from humans, a result that validates the utility of the method with large genomes and suggests that human potassium channel gene diversity may be extensive
— id: 18841, year: 1989, vol: 86, page: 4372, stat: Journal Article,

A FUNNEL-WEB SPIDER TOXIN FTX FRACTION BLOCKS CALCIUM CURRENTS INDUCED BY RAT BRAIN MESSENGER RNA IN XENOPUS OOCYTES
LIN J W; RUDY B; CHERKSEY B; SUGIMORI M; LLINAS R
1989 ;15(1):652-652, Abstracts (Society for Neuroscience)
— id: 92410, year: 1989, vol: 15, page: 652, stat: Journal Article,

FTX blocks calcium current expressed from rat brain mRNA in Xenopus oocytes
Lin JW; Rudy B; Llinas R
1989 ;177(2):323-323, Biological bulletin
— id: 55742, year: 1989, vol: 177, page: 323, stat: Journal Article,

CLONING AND CHARACTERIZATION OF HUMAN POTASSIUM CHANNEL GENES
MATHEW M K; RAMASWAMI M; GAUTAM M; KAMB C A; RUDY B; TANOUYE M A
1989 ;15(1):540-540, Abstracts (Society for Neuroscience)
— id: 92568, year: 1989, vol: 15, page: 540, stat: Journal Article,

SHAKER POTASSIUM CHANNELS A LEUCINE ZIPPER MOTIF MAY INDICATE A SITE FOR SUBUNIT INTERACTION AND GATING
MCCORMACK K; RAMASWAMI M; MATHEW M K; TANOUYE M A; IVERSON L; MCCORMACK T; RUDY B
1989 ;15(1):337-337, Abstracts (Society for Neuroscience)
— id: 92570, year: 1989, vol: 15, page: 337, stat: Journal Article,

LEUCINE-ZIPPER MOTIF UPDATE
Mccormack, K; Campanelli, JT; Ramaswami, M; Mathew, MK; Tanouye, MA; Iverson, LE; Rudy, B
1989 Jul 13;340(6229):103-103, Nature
— id: 31683, year: 1989, vol: 340, page: 103, stat: Journal Article,

GENERATION OF MULTIPLE POTASSIUM CHANNEL TYPES FROM THE DROSOPHILA-SHAKER (SH) LOCUS
Rudy, B; Iverson, LE; Tanouye, MA
1989 Sep 16;5(4):272-272, Journal of neurogenetics
— id: 31672, year: 1989, vol: 5, page: 272, stat: Journal Article,

ISOLATION OF A VOLTAGE-DEPENDENT CALCIUM CHANNEL FROM MAMMALIAN CNS
CHERKSEY B; SUGIMORI M; RUDY B; LLINAS L
1988 ;14(2):901-901, Abstracts (Society for Neuroscience)
— id: 92449, year: 1988, vol: 14, page: 901, stat: Journal Article,

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes
Farley J; Rudy B
1988 Jun;53(6):919-934, Biophysical journal
K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 18843, year: 1988, vol: 53, page: 919, stat: Journal Article,

A-type potassium channels expressed from Shaker locus cDNA
Iverson LE; Tanouye MA; Lester HA; Davidson N; Rudy B
1988 Aug;85(15):5723-5727, Proceedings of the National Academy of Sciences of the United States of America
A-type K+ currents are expressed in Xenopus oocytes injected with in vitro-synthesized transcripts from cDNAs for the Drosophila Shaker (Sh) locus. A single Sh gene product, possibly as a multimer, is sufficient for formation of functional A channels. Various Sh RNAs express A currents with distinct kinetic properties. An analysis of structure-function relationships shows that the conserved central region of Sh polypeptides determines ionic selectivity and overall channel behavior, whereas the divergent amino and carboxyl termini can modify channel kinetics. Alternative splicing of Sh gene transcripts may provide one mechanism for the generation of K+ channel diversity
— id: 18842, year: 1988, vol: 85, page: 5723, stat: Journal Article,

HUMAN AND BOVINE BEGF BUT NOT CYCLIC AMP INDUCE SODIUM CHANNELS IN PC12 PHEOCHROMOCYTOMA CELLS
POLLOCK J D; KREMPIN M; RUDY B
1988 ;14(1):141-141, Abstracts (Society for Neuroscience)
— id: 92572, year: 1988, vol: 14, page: 141, stat: Journal Article,

Diversity and ubiquity of K channels
Rudy B
1988 Jun;25(3):729-749, Neuroscience
— id: 11087, year: 1988, vol: 25, page: 729, stat: Journal Article,

At least two mRNA species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes
Rudy B; Hoger JH; Lester HA; Davidson N
1988 Oct;1(8):649-658, Neuron
Fast transient K+ channels (A channels) of the type operating in the subthreshold region for Na+ action potential generation were expressed in Xenopus oocytes injected with rat brain poly(A) RNA. Sucrose gradient fractionation of the RNA separates mRNAs encoding A-currents (6-7 kb) from mRNAs encoding other voltage-dependent K+ channels. A-currents expressed with fractionated mRNA differ in kinetics and pharmacology from A-currents expressed with total mRNA. The original properties of the A-currents can be reconstituted when small mRNAs (2-4 kb) are added to the large mRNA fraction. Thus the properties of the A-currents expressed with total poly(A) RNA depend on the presence of more than one mRNA species. mRNA(s) present in the large RNA fraction must encode channel subunits since they express an A-current by themselves. The small mRNA(s) may encode a second subunit(s) or a factor, such as an enzymatic activity that modulates the properties of the channels, which could play a role in generating A-channel functional diversity
— id: 10940, year: 1988, vol: 1, page: 649, stat: Journal Article,

LONG-TERM REGULATION OF POTASSIUM CHANNELS IN PC12 PHEOCHROMOCYTOMA CELLS BY NERVE GROWTH FACTOR
RUDY B; POLLOCK J D
1988 ;14(1):141-141, Abstracts (Society for Neuroscience)
— id: 92573, year: 1988, vol: 14, page: 141, stat: Journal Article,

AT LAST POTASSIUM CHANNELS EXPRESSION AND REGULATION
SPITZER N C; ADAMS P R; ALDRICH R W; JAN L Y; RUDY B
1988 ;14(1):300-300, Abstracts (Society for Neuroscience)
— id: 92571, year: 1988, vol: 14, page: 300, stat: Journal Article,

Effects of detergent on the binding of solubilized sodium channels to immobilized wheat germ agglutinin: structural implications
Weiner JS; Rudy B
1988 Oct 20;944(3):521-526, Biochimica & biophysica acta
The binding of the solubilized voltage-dependent sodium channel from rat brain to immobilized wheat germ agglutinin (WGA) is detergent-dependent. When solubilized in sodium cholate, only 11% of total recovered channels bound to a WGA-Sepharose column. When solubilized in Triton X-100 or CHAPS, however, 80% and 60% could bind, respectively. The effect of cholate on sodium channel binding is relatively specific: the major rat brain glycoproteins which bind to immobilized WGA are roughly the same in either Triton or cholate, as analyzed by SDS gel electrophoresis. The structural implications for the channel are discussed
— id: 10925, year: 1988, vol: 944, page: 521, stat: Journal Article,

DIVERSITY OF VOLTAGE DEPENDENT POTASSIUM CHANNELS INDUCED IN XENOPUS OOCYTES BY TOTAL AND FRACTIONATED RAT BRAIN MESSENGER RNA
HOGER J H; AHMED I; DAVIDSON N; LESTER H; RUDY B
1987 ;13(1):177-177, Abstracts (Society for Neuroscience)
— id: 92576, year: 1987, vol: 13, page: 177, stat: Journal Article,

INDUCTION OF SODIUM CHANNELS AND SODIUM CHANNEL MESSENGER RNA BY NGF IN PC12 CELLS
KIRSCHENBAUM B; SNUTCH T; LESTER H; GREENE L A; DAVIDSON N; RUDY B
1987 ;13(2):795-795, Abstracts (Society for Neuroscience)
— id: 92574, year: 1987, vol: 13, page: 795, stat: Journal Article,

EXPRESSION OF DIFFERENT A CURRENTS IN XENOPUS OOCYTES INJECTED WITH TOTAL OR FRACTIONATED RAT BRAIN MESSENGER RNA
RUDY B; HOGER J H; DAVIDSON N; LESTER H
1987 ;13(1):178-178, Abstracts (Society for Neuroscience)
— id: 92575, year: 1987, vol: 13, page: 178, stat: Journal Article,

Nerve growth factor increases the number of functional Na channels and induces TTX-resistant Na channels in PC12 pheochromocytoma cells
Rudy B; Kirschenbaum B; Rukenstein A; Greene LA
1987 Jun;7(6):1613-1625, Journal of neuroscience
The PC12 clone is a line of rat pheochromocytoma cells that undergoes neuronal differentiation in the presence of NGF protein. In the absence of NGF, PC12 cells are electrically inexcitable, while after several weeks of NGF treatment they develope Na+ action potentials. Past estimates made by measuring binding of 3H-saxitoxin (STX) indicate that NGF treatment brings about a large increase in Na channel density that is of sufficient magnitude to account for the induction of excitability. We have now used 22Na uptake to measure the Na permeability of PC12 cells before and after long-term NGF treatment. Treatment with NGF does not change the resting Na+ permeability. The alkaloid toxins veratridine and batrachotoxin (BTX) and scorpion toxin were used to activate Na channels. Such studies demonstrate that these toxins induce TTX-sensitive Na uptake in both NGF-treated and untreated cells and reveal differences in functional Na channel numbers per cell and per unit of membrane area that are similar to those found in the STX binding studies. On the other hand, affinities for drugs that activate these channels are not affected by NGF treatment. We also find that NGF-treated PC12 cells contain a population of Na channels with low affinity for TTX. These channels account for 5-20% of total BTX or veratridine-stimulated flux. Thus, NGF has 2 effects regarding the Na channels of PC12 cells: it increases the number of functional Na channels that otherwise behave similarly to those present before NGF treatment, and it induces the presence of TTX-resistant Na channels. These findings indicate that the PC12 model system may serve to study the developmental regulation of Na channel expression and properties
— id: 18844, year: 1987, vol: 7, page: 1613, stat: Journal Article,

Interactions between membranes and cytolytic peptides
Bernheimer AW; Rudy B
1986 Jun 12;864(1):123-141, Biochimica & biophysica acta
The physico-chemical and biological properties of cytolytic peptides derived from diverse living entities have been discussed. The principal sources of these agents are bacteria, higher fungi, cnidarians (coelenterates) and the venoms of snakes, insects and other arthropods. Attention has been directed to instances in which cytolytic peptides obtained from phylogenetically remote as well as from related sources show similarities in nature and/or mode of action (congeneric lysins). The manner in which cytolytic peptides interact with plasma membranes of eukaryotic cells, particularly the membranes of erythrocytes, has been discussed with emphasis on melittin, thiolactivated lysins and staphylococcal alpha-toxin. These and other lytic peptides are characterized in Table III. They can be broadly categorized into: (a) those which alter permeability to allow passage of ions, this process eventuating in colloid osmotic lysis, signs of which are a pre-lytic induction or latent period, pre-lytic leakage of potassium ions, cell swelling and inhibition of lysis by sucrose. Examples of lysins in which this mechanism is involved are staphylococcal alpha-toxin, streptolysin S and aerolysin; (b) phospholipases causing enzymic degradation of bilayer phospholipids as exemplified by phospholipases C of Cl. perfringens and certain other bacteria; (c) channel-forming agents such as helianthin, gramicidin and (probably) staphylococcal delta-toxin in which toxin molecules are thought to embed themselves in the membrane to form oligomeric transmembrane channels
— id: 18845, year: 1986, vol: 864, page: 123, stat: Journal Article,

SINGLE K+ CHANNELS FROM RAT AND HERMISSENDA BRAIN INCORPORATED INTO LIPID BILAYERS ON PATCH-CLAMP PIPETTES
FARLEY, J; RUDY, B
1986 FEB ;49(2):A575-A575, Biophysical journal
— id: 41508, year: 1986, vol: 49, page: A575, stat: Journal Article,

PROTEIN KINASE C EFFECTS ON SINGLE POTASSIUM CHANNELS FROM MAMMALIAN BRAIN
LIPKIN S; FARLEY J; RUDY B
1986 ;12(2):1343-1343, Abstracts (Society for Neuroscience)
— id: 92578, year: 1986, vol: 12, page: 1343, stat: Journal Article,

CYCLIC AMP DEPENDENT PROTEIN KINASE OPENS SEVERAL POTASSIUM CHANNELS FROM MAMMALIAN BRAIN
REEVES R; FARLEY J; RUDY B
1986 ;12(2):1343-1343, Abstracts (Society for Neuroscience)
— id: 92577, year: 1986, vol: 12, page: 1343, stat: Journal Article,

NERVE GROWTH FACTOR AND SODIUM CHANNEL DEVELOPMENT IN PC-12 CELLS
RUDY B; KIRSCHENBAUM B; GREENE L A
1986 ;12(2):950-950, Abstracts (Society for Neuroscience)
— id: 92579, year: 1986, vol: 12, page: 950, stat: Journal Article,

SINGLE POTASSIUM CHANNELS FROM RAT BRAIN INCORPORATED INTO LIPID BILAYERS ON PATCH-CLAMP PIPETTES
FARLEY J; RUDY B
1985 ;11(1):315-315, Abstracts (Society for Neuroscience)
— id: 92580, year: 1985, vol: 11, page: 315, stat: Journal Article,

SOLUBILIZATION WITH CHOLATE AND RECONSTITUTION OF THE NA+ CHANNEL FROM RAT-BRAIN
WEINER, JS; RUDY, B
1984 ;45(2):A288-A288, Biophysical journal
— id: 41023, year: 1984, vol: 45, page: A288, stat: Journal Article,

A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles
Garty H; Rudy B; Karlish SJ
1983 Nov 10;258(21):13094-13099, Journal of biological chemistry
In this paper, we describe a simple and highly sensitive manual assay for isotope fluxes through ion-conducting pathways, particularly cation-specific channels, in heterogenous populations of small membrane vesicles. We measure uptake of tracer of the ion of interest, against a large chemical gradient of the same ion. As a result of the imposed chemical gradient, a transient electrical diffusion potential is set up across the membranes of those vesicles which are highly permeable to the ion of interest. The isotope tends to equilibrate with the diffusion potential and is therefore concentrated selectively and transiently into those vesicle containing the channels. Furthermore, when performed in this way, the time course of tracer equilibration occurs over several minutes, rather than the sub-second range expected for tracer equilibration into channel-containing vesicles in the absence of an opposing chemical gradient of the permeant ion. The use of the procedure is demonstrated for three Na-conducting channels: gramicidin D incorporated into phospholipid vesicles, amiloride-blockable Na channels in toad bladder microsomes, and veratridine-activated tetrodotoxin-blockable Na channels in rat brain synaptic membranes. For all three cases, it proved simple to measure a specific 22Na uptake, in a minute time range, using very low concentrations of the channel-containing vesicles. By comparison with isotope flux measurements performed without an opposing Na gradient, the power of the present assay derives from both the very large gain in sensitivity and the convenient time course
— id: 18847, year: 1983, vol: 258, page: 13094, stat: Journal Article,

Formation and properties of cell-size lipid bilayer vesicles
Mueller P; Chien TF; Rudy B
1983 Dec;44(3):375-381, Biophysical journal
Hydration of single or mixed phospholipids or lipid protein mixtures at low ionic strength results in the formation of a population of large, solvent free, single bilayer vesicles with included volumes of up to 300 microliters/mumol lipid. Their size ranges from 0.1 to 300 microns and they can be sorted out according to size by centrifugation. When formed in distilled water their internal solution has a conductivity of 20-50 microseconds/cm-1, an osmolarity of 0.5-5 mOsM, and a density of 1.0005-1.001. The osmotic pressure produced by the internal solutes cause a surface stress of 25 dyn/cm for a 20-microns vesicle. Their elastic constant ranges from 75-150 dyn/cm. During formation they can internalize particles such as latex beads or cell nuclei. They can be impaled with microelectrodes, or patch clamped. They can also be sealed to a small Vaseline-treated hole in a thin partition between two aqueous compartments. Sealing occurs in two stages. In the first stage sealing resistance is similar to that seen with patch-clamp pipettes. In the second stage, a much tighter seal is obtained. After sealing, the smaller portion of the sealed vesicle can be selectively broken by an electric shock leaving a single membrane across the hole. The capacitance and resistance of such membranes, in the presence of 10 mM NaCl, are approximately 0.7 microF/cm2 and 10(8) omega cm2 for pure lipid vesicles. Gramicidin increases the membrane conductance and monazomycin induces voltage-dependent gating thus providing further evidence that the vesicles are bounded by a single bilayer
— id: 18846, year: 1983, vol: 44, page: 375, stat: Journal Article,

Nerve growth factor-induced increase in saxitoxin binding to rat PC12 pheochromocytoma cells
Rudy B; Kirschenbaum B; Greene LA
1982 Oct;2(10):1405-1411, Journal of neuroscience
The PC12 clone is a line of rat pheochromocytoma cells which undergoes neuronal differentiation in the presence of nerve growth factor (NGF) protein. In the absence of NGF, PC12 cells are electrically inexcitable, while after several weeks of NGF treatment, they develop sodium action potentials. The number and density of sodium channels on PC12 cells before and after treatment with NGF were estimated by measuring the binding of [3H]saxitoxin ([3H]STX). The data indicate that [3H]STX binding increases in the NGF-treated cells by 15- to 20-fold per cell, 3- to 10-fold per mg of protein, and an estimated 7-fold per unit area of membrane. The kinetic properties for [3H]STX binding are unchanged, however, by NGF treatment. A Hodgkin-Huxley analysis (Hodgkin, A. L., and A. F. Huxley (1952) J. Physiol. (Lond.) 117: 500-544) suggests that the estimated density of sodium channels in NGF-untreated PC12 cells is sufficient to explain their lack of excitability. On the other hand, the estimated channel density on the NGF-treated cells (30 to 50/micrometers 2) is comparable to that in other excitable systems. Thus, the development of excitability in PC12 cells in response to NGF could be due to the induction of sodium channel synthesis
— id: 18848, year: 1982, vol: 2, page: 1405, stat: Journal Article,

Inactivation in Myxicola giant axons responsible for slow and accumulative adaptation phenomena
Rudy B
1981 Mar;312(10):531-549, Journal of physiology
1. The action potential in Myxicola giant axons is abolished if the nerve is stimulated at frequencies higher than about 5 sec-1. At 1 sec-1 the magnitude of the action potential is not maintained upon sequential stimulation but decreases until the response is abolished. 2. The behaviour of the ionic currents underlying the action potential was studied with voltage-clamp techniques to find the origin of such adaptation. These studies showed a frequency-dependent decline of the sodium currents. 3. The decline in the Na currents upon repetitive depolarization is shown to be due to a decrease in the Na conductance and not to change in driving force. 4. An analysis of the effects of conditioning depolarizations on the Na current during a depolarizing test pulse demonstrates that in a single short depolarization (less than 10 msec) 15% of the Na conductance enters an inactivated state from which recovery is very slow. Upon repetitive depolarizations the amount of Na conductance available accumulates in this slowly recovering inactivated state. 5. The data are explained by proposing that every time the membrane is depolarized open channels undergo one of two competing reactions. Open channels enter either the traditional inactivated state described by Hodgkin & Huxley (1952b) from which recovery is fast (a few milliseconds) or an inactivated state from which recovery is very slow (seconds). In Myxicola, only 15% of open channels enter the later inactivated state in a single depolarization. Upon repetitive depolarizations, however, the fraction in this state accumulates if the frequency of pulsing is faster than the rate of recovery. 6. Axons in which the amount of open channels entering the slowly recovering inactivated state is significant, such as in Myxicola, have thus a system capable of storing the previous activity of the axon for periods of seconds or minutes
— id: 18849, year: 1981, vol: 312, page: 531, stat: Journal Article,

Arginine-specific reagents remove sodium channel inactivation
Eaton DC; Brodwick MS; Oxford GS; Rudy B
1978 Feb 2;271(5644):473-476, Nature
— id: 18851, year: 1978, vol: 271, page: 473, stat: Journal Article,

Slow inactivation of the sodium conductance in squid giant axons. Pronase resistance
Rudy B
1978 Oct;283(10):1-21, Journal of physiology
1. Squid giant axons internally perfused with CsF have their Na conductance inactivated due to the low value of the resting potential. When hyperpolarized with voltage clamp to normal values of resting potential, the Na conductance recovers with an exponential time course. The time constant of recovery is of the order of 30 sec at a membrane potential of -70 mV and at 5 degrees C. The recovery from slow inactivation has a Q10 of about 3. 2. The development of inactivation during depolarization is also slow. The time constant varies between 10 and 20 sec at 5 degrees C, depending upon the value of the membrane potential. 3. Slow inactivation is also observed in NaF perfused axons and in intact axons with a low resting potential. 4. Although internal perfusion with pronase (or a purified fraction of this enzymic complex) blocks the fast (h) inactivation of the Na conductance, the slow inactivation remains. The recovery is similar before and after the proteolytic treatment. However, slow inactivation appears to develop faster after enzymic perfusion. 5. Slow inactivation develops without any apparent change in distributed or local membrane surface charge. 6. The experiments suggest that slow inactivation is a general property of the Na conductance as in many other conductance channels in excitable membranes. The experiments can be interpreted by proposing that slow inactivation is a phenomenon independent of fast inactivation, and that pronase somehow accelerates the onset of slow inactivation. 7. An alternative model, in which slow inactivation is coupled to fast inactivation, is proposed. This model is consistent with the results presented here and is very similar to one proposed to explain the frequency response of the sodium currents in Myxicola giant axons (Rudy, 1975, 1978)
— id: 18850, year: 1978, vol: 283, page: 1, stat: Journal Article,

Destruction of the sodium conductance inactivation by a specific protease in perfused nerve fibres from Loligo
Rojas E; Rudy B
1976 Nov;262(2):501-531, Journal of physiology
Intracellular perfusion of giant axons from Loligo forbesi with a crude protein extract of Pronase dissolved in a KF solution suppresses the process of fast inactivation of the Na conductance (the h-process in the Hodgkin-Huxley terminology). 2. The results with protease inhibitors indicate that the most substrate specific endopeptidase present in pronase, alkaline proteinase b, destroys the h-process. 3. After destruction of the inactivation the conductance rise upon depolarization followed cube law kinetics. Values of the time constant taum before and after destruction of the h-process were very similar. 4. After destruction of the inactivation process the following properties were tested: cation selectivity, instantaneous conductance and internal receptor sites for tetrodotoxin (TTX) and tetraethylammonium (TEA). No detectable changes in selectivity or instantaneous conductance were observed. No internal receptors for TTX affecting the Na conductance were found but a TEA receptor is exposed by the protein hydrolysis. 5. TEA derivatives (triethylammonium, TEA-, with an aliphatic chain, Cn) induce a partial block of the steady-state sodium current and induce a time-dependent blockage of the conductance. 6. The first effect of TEA-Cn could be described in terms of a unimolecular reaction with the following equilibrium constants: 50, 2-5, 1-0, 0-4 and 0-025 mM for TEA-C2, TEA-C4, TEA-C5, TEA-C7 and TEA-C9 respectively. 7. From the dependence of the equilibrium dissociation constant on the length of the alkyl chain we estimated the free-energy change in 560 cal/mole of CH2. The gain in free energy per CH2 group transferred from aqueous medium to the interior of a non-polar medium is 1000 cal. 8. Although with the data at hand it is impossible to propose the amino-acid sequence of the site cleaved by alkaline proteinase b, we propose that an important functional component is arginine (or lysine)
— id: 18852, year: 1976, vol: 262, page: 501, stat: Journal Article,

Sodium gating currents in Myxicola giant axons
Rudy B
1976 Jun 30;193(1113):469-475, Proceedings of the Royal Society. B. Biological sciences
— id: 18853, year: 1976, vol: 193, page: 469, stat: Journal Article,

Proceedings: Slow recovery of the inactivation of sodium conductance in Myxicola giant axons
Rudy B
1975 Jul;249(1):22P-24P, Journal of physiology
— id: 18855, year: 1975, vol: 249, page: 22P, stat: Journal Article,

A pharmacologically active derivative of tetrodotoxin
Tsien RY; Green DP; Levinson SR; Rudy B; Sanders JK
1975 Dec 16;191(1105):555-559, Proceedings of the Royal Society. B. Biological sciences
— id: 18854, year: 1975, vol: 191, page: 555, stat: Journal Article,

Proceedings: Demonstration of a first-order voltage-dependent transition of the sodium activation gates
Keynes RD; Rojas E; Rudy B
1974 Jun;239(2):100P-101P, Journal of physiology
— id: 18856, year: 1974, vol: 239, page: 100P, stat: Journal Article,

Microviscosity of the cell membrane
Rudy B; Gitler C
1972 Oct 23;288(1):231-236, Biochimica & biophysica acta
— id: 18857, year: 1972, vol: 288, page: 231, stat: Journal Article,

Hereditary perpetuation of a membrane state in the absence of genic alterations. A hypothesis
Gitler, C; Rudy, B
1971 Oct;27(4):113-117, Boletin de estudios medicos y biologicos
— id: 18858, year: 1971, vol: 27, page: 113, stat: Journal Article,