William A Coetzee

Lethal arrhythmias in Tbx3-deficient mice reveal extreme dosage sensitivity of cardiac conduction system function and homeostasis
Frank, Deborah U.; Carter, Kandis L.; Thomas, Kirk R.; Burr, R. Michael; Bakker, Martijn L.; Coetzee, William A.; Tristani-Firouzi, Martin; Bamshad, Michael J.; Christoffels, Vincent M.; Moon, Anne M.
2012 JAN 17 ;109(3):E154-E163, Proceedings of the National Academy of Sciences of the United States of America
TBX3 is critical for human development: mutations in TBX3 cause congenital anomalies in patients with ulnar-mammary syndrome. Data from mice and humans suggest multiple roles for Tbx3 in development and function of the cardiac conduction system. The mechanisms underlying the functional development, maturation, and maintenance of the conduction system are not well understood. We tested the requirements for Tbx3 in these processes. We generated a unique series of Tbx3 hypomorphic and conditional mouse mutants with varying levels and locations of Tbx3 activity within the heart, and developed techniques for evaluating in vivo embryonic conduction system function. Disruption of Tbx3 function in different regions of the developing heart causes discrete phenotypes and lethal arrhythmias: sinus pauses and bradycardia indicate sinoatrial node dysfunction, whereas preexcitation and atrioventricular block reveal abnormalities in the atrioventricular junction. Surviving Tbx3 mutants are at increased risk for sudden death. Arrhythmias induced by knockdown of Tbx3 in adults reveal its requirement for conduction system homeostasis. Arrhythmias in Tbx3-deficient embryos are accompanied by disrupted expression of multiple ion channels despite preserved expression of previously described conduction system markers. These findings indicate that Tbx3 is required for the conduction system to establish and maintain its correct molecular identity and functional properties. In conclusion, Tbx3 is required for the functional development, maturation, and homeostasis of the conduction system in a highly dosage-sensitive manner. TBX3 and its regulatory targets merit investigation as candidates for human arrhythmias
— id: 150786, year: 2012, vol: 109, page: E154, stat: Journal Article,

AMP-activated protein kinase connects cellular energy metabolism to K(ATP) channel function
Yoshida, Hidetada; Bao, Li; Kefaloyianni, Eirini; Taskin, Eylem; Okorie, Uzoma; Hong, Miyoun; Dhar-Chowdhury, Piyali; Kaneko, Michiyo; Coetzee, William A
2012 Feb;52(2):410-418, Journal of molecular & cellular cardiology
AMPK is an important sensor of cellular energy levels. The aim of these studies was to investigate whether cardiac K(ATP) channels, which couple cellular energy metabolism to membrane excitability, are regulated by AMPK activity. We investigated effects of AMPK on rat ventricular K(ATP) channels using electrophysiological and biochemical approaches. Whole-cell K(ATP) channel current was activated by metabolic inhibition; this occurred more rapidly in the presence of AICAR (an AMPK activator). AICAR had no effects on K(ATP) channel activity recorded in the inside-out patch clamp configuration, but ZMP (the intracellular intermediate of AICAR) strongly activated K(ATP) channels. An AMPK-mediated effect is demonstrated by the finding that ZMP had no effect on K(ATP) channels in the presence of Compound C (an AMPK inhibitor). Recombinant AMPK activated Kir6.2/SUR2A channels in a manner that was dependent on the AMP concentration, whereas heat-inactivated AMPK was without effect. Using mass-spectrometry and co-immunoprecipitation approaches, we demonstrate that the AMPK alpha-subunit physically associates with K(ATP) channel subunits. Our data demonstrate that the cardiac K(ATP) channel function is directly regulated by AMPK activation. During metabolic stress, a small change in cellular AMP that activates AMPK can be a potential trigger for K(ATP) channel opening. This article is part of a Special Issue entitled 'Local Signaling in Myocytes'
— id: 150558, year: 2012, vol: 52, page: 410, stat: Journal Article,

Endosomal KATP channels as a reservoir after myocardial ischemia: a role for SUR2 subunits
Bao, Li; Hadjiolova, Krassimira; Coetzee, William A; Rindler, Michael J
2011 Jan;300(1):H262-H270, American journal of physiology. Heart & circulatory physiology
ATP-sensitive K(+) (K(ATP)) channels, composed of inward rectifier K(+) (Kir)6.x and sulfonylurea receptor (SUR)x subunits, are expressed on cellular plasma membranes. We demonstrate an essential role for SUR2 subunits in trafficking K(ATP) channels to an intracellular vesicular compartment. Transfection of Kir6.x/SUR2 subunits into a variety of cell lines (including h9c2 cardiac cells and human coronary artery smooth muscle cells) resulted in trafficking to endosomal/lysosomal compartments, as assessed by immunofluorescence microscopy. By contrast, SUR1/Kir6.x channels efficiently localized to the plasmalemma. The channel turnover rate was similar with SUR1 or SUR2, suggesting that the expression of Kir6/SUR2 proteins in lysosomes is not associated with increased degradation. Surface labeling of hemagglutinin-tagged channels demonstrated that SUR2-containing channels dynamically cycle between endosomal and plasmalemmal compartments. In addition, Kir6.2 and SUR2 subunits were found in both endosomal and sarcolemmal membrane fractions isolated from rat hearts. The balance of these K(ATP) channel subunits shifted to the sarcolemmal membrane fraction after the induction of ischemia. The K(ATP) channel current density was also increased in rat ventricular myocytes isolated from hearts rendered ischemic before cell isolation without corresponding changes in subunit mRNA expression. We conclude that an intracellular pool of SUR2-containing K(ATP) channels exists that is derived by endocytosis from the plasma membrane. In cardiac myocytes, this pool can potentially play a cardioprotective role by serving as a reservoir for modulating surface K(ATP) channel density under stress conditions, such as myocardial ischemia
— id: 117338, year: 2011, vol: 300, page: H262, stat: Journal Article,

Unique Properties of the ATP-Sensitive K+ Channel in the Mouse Ventricular Cardiac Conduction System
Bao, Li; Kefaloyianni, Eirini; Lader, Joshua; Hong, Miyoun; Morley, Gregory; Fishman, Glenn I; Sobie, Eric A; Coetzee, William A
2011 Dec 1;4(6):926-935, Circulation. Arrhythmia & electrophysiology
Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia
— id: 148727, year: 2011, vol: 4, page: 926, stat: Journal Article,

The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load
Chin, King-Tung; Kang, Guoxin; Qu, Jiaxiang; Gardner, Lawrence B; Coetzee, William A; Zito, Ester; Fishman, Glenn I; Ron, David
2011 Aug;25(8):2583-2591, FASEB journal
Two related ER oxidation 1 (ERO1) proteins, ERO1alpha and ERO1beta, dynamically regulate the redox environment in the mammalian endoplasmic reticulum (ER). Redox changes in cysteine residues on intralumenal loops of calcium release and reuptake channels have been implicated in altered calcium release and reuptake. These findings led us to hypothesize that altered ERO1 activity may affect cardiac functions that are dependent on intracellular calcium flux. We established mouse lines with loss of function insertion mutations in Ero1l and Ero1lb encoding ERO1alpha and ERO1beta. The peak amplitude of calcium transients in homozygous Ero1alpha mutant adult cardiomyocytes was reduced to 42.0 +/- 2.2% (n=10, P</=0.01) of values recorded in wild-type cardiomyocytes. Decreased ERO1 activity blunted cardiomyocyte inotropic response to adrenergic stimulation and sensitized mice to adrenergic blockade. Whereas all 12 wild-type mice survived challenge with 4 mg/kg esmolol, 6 of 8 compound Ero1l and Ero1lb mutant mice succumbed to this level of beta adrenergic blockade (P</=0.01). In addition, mice lacking ERO1alpha were partially protected against progressive heart failure in a transaortic constriction model [at 10 wk postprocedure, fractional shortening was 0.31+/-0.02 in the mutant (n=20) vs. 0.23+/-0.03 in the wild type (n=18); P</=0.01]. These findings establish a role for ERO1 in calcium homeostasis and suggest that modifying the lumenal redox environment may affect the progression of heart failure.-Chin, K. T., Kang, G., Qu, J., Gardner, L. B., Coetzee, W. A., Zito, E., Fishman, G. I., Ron, R. The sarcoplasmic reticulum luminal thiol oxidase ERO1 regulates cardiomyocyte excitation-coupled calcium release and response to hemodynamic load
— id: 135555, year: 2011, vol: 25, page: 2583, stat: Journal Article,

Cardiac ATP-sensitive K+ channel associates with the glycolytic enzyme complex
Hong, Miyoun; Kefaloyianni, Eirini; Bao, Li; Malester, Brian; Delaroche, Diane; Neubert, Thomas A; Coetzee, William A
2011 Jul;25(7):2456-2467, FASEB journal
Being gated by high-energy nucleotides, cardiac ATP-sensitive potassium (K(ATP)) channels are exquisitely sensitive to changes in cellular energy metabolism. An emerging view is that proteins associated with the K(ATP) channel provide an additional layer of regulation. Using putative sulfonylurea receptor (SUR) coiled-coil domains as baits in a 2-hybrid screen against a rat cardiac cDNA library, we identified glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and SUR was confirmed using GST pulldown assays and coimmunoprecipitation assays. Mass spectrometry of proteins from K(ATP) channel immunoprecipitates of rat cardiac membranes identified glycolysis as the most enriched biological process. Coimmunoprecipitation assays confirmed interaction for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with K(ATP) channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate K(ATP) channels in patch-clamp experiments, whereas d-glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP-generating steps) with cardiac K(ATP) channels.-Hong, M., Kefaloyianni, E., Bao, L., Malester, B., Delaroche, D., Neubert, T. A., Coetzee, W. A. Cardiac ATP-sensitive K(+) channel associates with the glycolytic enzyme complex
— id: 134908, year: 2011, vol: 25, page: 2456, stat: Journal Article,

Transcriptional remodeling of ion channel subunits by flow adaptation in human coronary artery endothelial cells
Kefaloyianni, Eirini; Coetzee, William A
2011 ;48(4):357-367, Journal of vascular research
Endothelial cells (ECs) are constantly exposed to blood flow-induced shear forces in the vessels and this is a major determinant of endothelial function. Ion channels have a major role in endothelial function and in the control of vascular tone. We hypothesized that shear force is a general regulator of ion channel expression, which will have profound effects on endothelial function. We examined this hypothesis using large-scale quantitative real-time RT-PCR. Human coronary artery ECs were exposed to two levels of flow-induced shear stress for 24 h, while control cells were grown under static conditions. The expression of ion channel subunits was compared between control and flow-adapted cells. We used primers against 55 ion channel and exchanger subunits and were able to detect 54 subunits. Five dyn/cm(2) of shear induced downregulation of 1 (NCX1) and upregulation of 18 subunits, including K(Ca)2.2, K(Ca)2.3, CX37, K(v)1.5 and HCN2. Fifteen dyn/cm(2) of shear stress induced the expression of 30 ion channel subunits, including K(Ca)2.3, K(Ca)2.2, CX37, K(ir)2.3 and K(Ca)3.1. Our data demonstrate that substantial remodeling of endothelial ion channel subunit expression occurs with flow adaptation and suggest that altered ion channel expression may significantly contribute to vascular pathology associated with flow-induced alterations
— id: 134437, year: 2011, vol: 48, page: 357, stat: Journal Article,

Remodeling of Atrial ATP-Sensitive Potassium Channels in a Model of Salt-induced Elevated Blood Pressure
Lader JM; Vasquez C; Bao L; Maass K; Qu J; Kefalogianni E; Fishman G; Coetzee WA; Morley GE
2011 Sep;301(3):H964-H974, American journal of physiology. Heart & circulatory physiology
Background: Hypertension is associated with the development of atrial fibrillation, however the electrophysiological consequences of this condition remain poorly understood. K(ATP) channels, which contribute to ventricular arrhythmias, are also expressed in the atria. We hypothesized that salt-induced elevated blood pressure leads to atrial K(ATP) channel activation and increased arrhythmia inducibility. Methods and Results: Elevated blood pressure was induced in mice with a high salt diet (HS) for four weeks. High resolution optical mapping was used to measure atrial arrhythmia inducibility, effective refractory period (ERP) and action potential duration (APD(90)). Excised patch clamping was performed to quantify K(ATP) channel properties and density. K(ATP) channel protein expression was also evaluated. Atrial arrhythmia inducibility was 22% higher in HS compared to control hearts. ERP and APD(90) were significantly shorter in the RAA and LAA of HS compared to control hearts. Perfusion with 1 muM glibenclamide or 300 muM tolbutamide significantly decreased arrhythmia inducibility and prolonged APD(90) in HS hearts compared to untreated HS hearts. K(ATP) channel density was 156% higher in myocytes isolated from HS compared to control animals. SUR1 protein expression was increased in the HS LAA (415% of NS) and RAA (372% of NS). Conclusion: K(ATP) channel activation provides a mechanistic link between salt-induced elevated BP and increased atrial arrhythmia inducibility. The findings of this study have important implications for the treatment and prevention of atrial arrhythmias in the setting of hypertensive heart disease and may lead to new therapeutic approaches
— id: 135528, year: 2011, vol: 301, page: H964, stat: Journal Article,

Short communication: flecainide exerts an antiarrhythmic effect in a mouse model of catecholaminergic polymorphic ventricular tachycardia by increasing the threshold for triggered activity
Liu, Nian; Denegri, Marco; Ruan, Yanfei; Avelino-Cruz, Jose Everardo; Perissi, Andrea; Negri, Sara; Napolitano, Carlo; Coetzee, William A; Boyden, Penelope A; Priori, Silvia G
2011 Jul 22;109(3):291-295, Circulation research
Rationale: Flecainide prevents arrhythmias in catecholaminergic polymorphic ventricular tachycardia, but the antiarrhythmic mechanism remains unresolved. It is possible for flecainide to directly affect the cardiac ryanodine receptor (RyR2); however, an extracellular site of action is suggested because of the hydrophilic nature of flecainide. Objective: To investigate the mechanism for the antiarrhythmic action of flecainide in a RyR2(R4496C+/-) knock-in mouse model of catecholaminergic polymorphic ventricular tachycardia. Methods and Results: Flecainide prevented catecholamine-induced sustained ventricular tachycardia in RyR2(R4496C+/-) mice. Cellular studies were performed with isolated RyR2(R4496C+/-) myocytes. Isoproterenol caused the appearance of spontaneous Ca(2+) transients, which were unaffected by flecainide (6 mumol/L). Flecainide did not affect Ca(2+) transient amplitude, decay, or sarcoplasmic reticulum Ca(2+) content. Moreover, it did not affect the frequency of spontaneous Ca(2+) sparks in permeabilized myocytes. In contrast, flecainide effectively prevented triggered activity induced by isoproterenol. The threshold for action potential induction was increased significantly (P<0.01), which suggests a primary extracellular antiarrhythmic effect mediated by Na(+) channel blockade. Conclusions: Flecainide prevents catecholaminergic polymorphic ventricular tachycardia in RyR2(R4496C+/-) mice; however, at variance with previous reports, we observed minimal effects on intracellular Ca(2+) homeostasis. Our data suggest that the antiarrhythmic activity of the drug is caused by reduction of Na(+) channel availability and by an increase in the threshold for triggered activity
— id: 135573, year: 2011, vol: 109, page: 291, stat: Journal Article,

Calmodulin kinase II inhibition prevents arrhythmias in RyR2(R4496C+/-) mice with catecholaminergic polymorphic ventricular tachycardia
Liu, Nian; Ruan, Yanfei; Denegri, Marco; Bachetti, Tiziana; Li, Yang; Colombi, Barbara; Napolitano, Carlo; Coetzee, William A; Priori, Silvia G
2011 Jan;50(1):214-222, Journal of molecular & cellular cardiology
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease characterized by life-threatening arrhythmias elicited by adrenergic activation. CPVT is caused by mutations in the cardiac ryanodine receptor gene (RyR2). In vitro studies demonstrated that RyR2 mutations respond to sympathetic activation with an abnormal diastolic Ca(2+) leak from the sarcoplasmic reticulum; however the pathways that mediate the response to adrenergic stimulation have not been defined. In our RyR2(R4496C+/-) knock-in mouse model of CPVT we tested the hypothesis that inhibition of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) counteracts the effects of adrenergic stimulation resulting in an antiarrhythmic activity. CaMKII inhibition with KN-93 completely prevented catecholamine-induced sustained ventricular tachyarrhythmia in RyR2(R4496C+/-) mice, while the inactive congener KN-92 had no effect. In ventricular myocytes isolated from the hearts of RyR2(R4496C+/-) mice, CaMKII inhibition with an autocamtide-2 related inhibitory peptide or with KN-93 blunted triggered activity and transient inward currents induced by isoproterenol. Isoproterenol also enhanced the activity of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), increased spontaneous Ca(2+) release and spark frequency. CaMKII inhibition blunted each of these parameters without having an effect on the SR Ca(2+) content. Our data therefore indicate that CaMKII inhibition is an effective intervention to prevent arrhythmogenesis (both in vivo and in vitro) in the RyR2(R4496C+/-) knock-in mouse model of CPVT. Mechanistically, CAMKII inhibition acts on several elements of the EC coupling cascade, including an attenuation of SR Ca(2+) leak and blunting catecholamine-mediated SERCA activation. CaMKII inhibition may therefore represent a novel therapeutic target for patients with CPVT
— id: 138269, year: 2011, vol: 50, page: 214, stat: Journal Article,

Subsecond regulation of striatal dopamine release by pre-synaptic K(ATP) channels
Patel, Jyoti C; Witkovsky, Paul; Coetzee, William A; Rice, Margaret E
2011 Sep;118(5):721-736, Journal of neurochemistry
J. Neurochem. (2011) 118, 721-736. ABSTRACT: ATP-sensitive K(+) (K(ATP) ) channels are composed of pore-forming subunits, typically Kir6.2 in neurons, and regulatory sulfonylurea receptor subunits. In dorsal striatum, activity-dependent H(2) O(2) produced from glutamate receptor activation inhibits dopamine release via K(ATP) channels. Sources of modulatory H(2) O(2) include striatal medium spiny neurons, but not dopaminergic axons. Using fast-scan cyclic voltammetry in guinea-pig striatal slices and immunohistochemistry, we determined the time window for H(2) O(2) /K(ATP) -channel-mediated inhibition and assessed whether modulatory K(ATP) channels are on dopaminergic axons. Comparison of paired-pulse suppression of dopamine release in the absence and presence of glibenclamide, a K(ATP) -channel blocker, or mercaptosuccinate, a glutathione peroxidase inhibitor that enhances endogenous H(2) O(2) levels, revealed a time window for inhibition of 500-1000 ms after stimulation. Immunohistochemistry demonstrated localization of Kir6.2 K(ATP) -channel subunits on dopaminergic axons. Consistent with the presence of functional K(ATP) channels on dopaminergic axons, K(ATP) -channel openers, diazoxide and cromakalim, suppressed single-pulse evoked dopamine release. Although cholinergic interneurons that tonically regulate dopamine release also express K(ATP) channels, diazoxide did not induce the enhanced frequency responsiveness of dopamine release seen with nicotinic-receptor blockade. Together, these studies reveal subsecond regulation of striatal dopamine release by endogenous H(2) O(2) acting at K(ATP) channels on dopaminergic axons, including a role in paired-pulse suppression
— id: 136636, year: 2011, vol: 118, page: 721, stat: Journal Article,

Reduction in number of sarcolemmal K(ATP) channels slows cardiac action potential duration shortening under hypoxia
Zhu, Zhiyong; Burnett, Colin M-L.; Maksymov, Gennadiy; Stepniak, Elizabeth; Sierra, Ana; Subbotina, Ekaterina; Anderson, Mark E.; Coetzee, William A.; Hodgson-Zingman, Denice M.; Zingman, Leonid V.
2011 DEC 2 ;415(4):637-641, Biochemical & biophysical research communications
The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (K(ATP)) channels. K(ATP) channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative K(ATP) channel subunit were compared with littermate controls. Evaluation of K(ATP) channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80-85% reduction in cardiac K(ATP) channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal K(ATP) channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress. (C) 2011 Elsevier Inc. All rights reserved
— id: 149883, year: 2011, vol: 415, page: 637, stat: Journal Article,

Exercise-induced expression of cardiac ATP-sensitive potassium channels promotes action potential shortening and energy conservation
Zingman, Leonid V; Zhu, Zhiyong; Sierra, Ana; Stepniak, Elizabeth; Burnett, Colin M-L; Maksymov, Gennadiy; Anderson, Mark E; Coetzee, William A; Hodgson-Zingman, Denice M
2011 Jul;51(1):72-81, Journal of molecular & cellular cardiology
Physical activity is one of the most important determinants of cardiac function. The ability of the heart to increase delivery of oxygen and metabolic fuels relies on an array of adaptive responses necessary to match bodily demand while avoiding exhaustion of cardiac resources. The ATP-sensitive potassium (K(ATP)) channel has the unique ability to adjust cardiac membrane excitability in accordance with ATP and ADP levels, and up-regulation of its expression that occurs in response to exercise could represent a critical element of this adaption. However, the mechanism by which K(ATP) channel expression changes result in a beneficial effect on cardiac excitability and function remains to be established. Here, we demonstrate that an exercise-induced rise in K(ATP) channel expression enhanced the rate and magnitude of action potential shortening in response to heart rate acceleration. This adaptation in membrane excitability promoted significant reduction in cardiac energy consumption under escalating workloads. Genetic disruption of normal K(ATP) channel pore function abolished the exercise-related changes in action potential duration adjustment and caused increased cardiac energy consumption. Thus, an expression-driven enhancement in the K(ATP) channel-dependent membrane response to alterations in cardiac workload represents a previously unrecognized mechanism for adaptation to physical activity and a potential target for cardioprotection
— id: 136465, year: 2011, vol: 51, page: 72, stat: Journal Article,

Sarcolemmal ATP-sensitive K(+) channels control energy expenditure determining body weight
Alekseev, Alexey E; Reyes, Santiago; Yamada, Satsuki; Hodgson-Zingman, Denice M; Sattiraju, Srinivasan; Zhu, Zhiyong; Sierra, Ana; Gerbin, Marina; Coetzee, William A; Goldhamer, David J; Terzic, Andre; Zingman, Leonid V
2010 Jan;11(1):58-69, Cell metabolism
Metabolic processes that regulate muscle energy use are major determinants of bodily energy balance. Here, we find that sarcolemmal ATP-sensitive K(+) (K(ATP)) channels, which couple membrane excitability with cellular metabolic pathways, set muscle energy expenditure under physiological stimuli. Disruption of K(ATP) channel function provoked, under conditions of unaltered locomotor activity and blood substrate availability, an extra energy cost of cardiac and skeletal muscle performance. Inefficient fuel metabolism in K(ATP) channel-deficient striated muscles reduced glycogen and fat body depots, promoting a lean phenotype. The propensity to lesser body weight imposed by K(ATP) channel deficit persisted under a high-fat diet, yet obesity restriction was achieved at the cost of compromised physical endurance. Thus, sarcolemmal K(ATP) channels govern muscle energy economy, and their downregulation in a tissue-specific manner could present an antiobesity strategy by rendering muscle increasingly thermogenic at rest and less fuel efficient during exercise
— id: 134977, year: 2010, vol: 11, page: 58, stat: Journal Article,

Novel insights into hydrogen sulfide--mediated cytoprotection
Calvert, John W; Coetzee, William A; Lefer, David J
2010 May 15;12(10):1203-1217, Antioxidants & Redox Signaling
Hydrogen sulfide (H(2)S) is a colorless, water soluble, flammable gas that has the characteristic smell of rotten eggs. Like other members of the gasotransmitter family (nitric oxide and carbon monoxide), H(2)S has traditionally been considered to be a highly toxic gas and environmental hazard. However, much like for nitric oxide and carbon monoxide, the initial negative perception of H(2)S has evolved with the discovery that H(2)S is produced enzymatically in mammals under normal conditions. As a result of this discovery, there has been a great deal of work to elucidate the physiological role of H(2)S. H(2)S is now recognized to be cytoprotective in various models of cellular injury. Specifically, it has been demonstrated that the acute administration of H(2)S, either prior to ischemia or at reperfusion, significantly ameliorates in vitro or in vivo myocardial and hepatic ischemia-reperfusion injury. These studies have also demonstrated a cardioprotective role for endogenous H(2)S. This review article summarizes the current body of evidence demonstrating the cytoprotective effects of H(2)S with an emphasis on the cardioprotective effects. This review also provides a detailed description of the current signaling mechanisms shown to be responsible for these cardioprotective actions
— id: 134344, year: 2010, vol: 12, page: 1203, stat: Journal Article,

Redox Regulation of ATP-sensitive Potassium Channels: A Possible Role for Thioredoxins
Bao, L; Delaroche, D; Malester, B; Coetzee, W
2009 NOV 3 ;120(18):S1070-S1070, Circulation
— id: 106983, year: 2009, vol: 120, page: S1070, stat: Journal Article,

Developmental programming resulting from maternal obesity in mice: effects on myocardial ischaemia-reperfusion injury
Calvert, JW; Lefer, DJ; Gundewar, S; Poston, L; Coetzee, WA
2009 JUL 1 ;94(7):805-814, Experimental physiology
A comprehensive number of epidemiological and animal studies suggest that prenatal and early life events are important determinants for disorders later in life. Among them, prenatal stress (i.e. stress experienced by the pregnant mother with impact on the fetal ontogeny) has clear programming effects on the cardiovascular system. A fetus developing in adverse conditions becomes an adult who is susceptible to disease, which may include hypertension, insulin resistance, altered blood lipid levels and cardiovascular disease. Recent evidence demonstrates that maternal programming can occur in the absence of other adverse environmental factors. Obesity, which is becoming a problem of large proportions in Western countries, is a possible cause of programming. With over 30% of the population of the USA currently obese, many mothers suffer from obesity during their child-bearing years (in fact, these conditions are often aggravated during pregnancy). One of the targets of programming is the cardiovascular system, and reported consequences include hypertension, endothelial dysfunction and vascular abnormalities. The overall goal of our study was to investigate the susceptibility of the heart to ischaemia-reperfusion in an animal model of maternal obesity. Our data demonstrate that normal (non-mutant) offspring from obese agouti mouse dams had an increased susceptibility to ischaemia-reperfusion injury. These data may provide insights into the long-term cardiovascular consequences of programming
— id: 100528, year: 2009, vol: 94, page: 805, stat: Journal Article,

SRp38 Regulates Alternative Splicing and Is Required for Ca2+ Handling in the Embryonic Heart
Feng, Y; Valley, MT; Lazar, J; Yang, AL; Bronson, RT; Firestein, S; Coetzee, WA; Manley, JL
2009 APR 21 ;16(4):528-538, Developmental cell
SRp38 is an atypical SR protein splicing regulator. To define the functions of SRp38 in vivo, we generated SRp38 null mice. The majority of homozygous mutants survived only until E15.5 and displayed multiple cardiac defects. Evaluation of gene expression profiles in the SRp38(-/-) embryonic heart revealed a defect in processing of the pre-mRNA encoding cardiac triadin, a protein that functions in regulation of Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling. This defect resulted in significantly reduced levels of triadin, as well as those of the interacting protein calsequestrin 2. Purified SRp38 was shown to bind specifically to the regulated exon and to modulate triadin splicing in vitro. Extending these results, isolated SRp38(-/-) embryonic cardiomyocytes displayed defects in Ca2+ handling compared with wild-type controls. Taken together, our results demonstrate that SRp38 regulates cardiac-specific alternative splicing of triadin pre-mRNA and, reflecting this, is essential for proper Ca2+ handling during embryonic heart development
— id: 97974, year: 2009, vol: 16, page: 528, stat: Journal Article,

Sulfonylurea receptor 1 subunits of ATP-sensitive potassium channels and myocardial ischemia/reperfusion injury
Lefer, David J; Nichols, Colin G; Coetzee, William A
2009 Feb;19(2):61-67, Trends in cardiovascular medicine
K(ATP) channels are generally cardioprotective under conditions of metabolic impairment, consisting of pore-forming (Kir6.1 and/or Kir6.2) and sulphonylurea-binding, modulatory subunits [sulfonylurea receptor (SUR) 1, 2A, or 2B]. Cardiovascular K(ATP) channels are generally thought to consist of Kir6.2/SUR2A subunits (in the case of heart muscle) or Kir6.1/SUR2B subunits (smooth muscle), whereas SUR1-containing channels have well-documented roles in pancreatic insulin release. Recent data, however, demonstrated the presence of SUR1 subunits in mouse cardiac tissue (particularly in atria) and a surprising protection from myocardial ischemia/reperfusion in SUR1-null mice. Here, we review some of the extra-pancreatic roles assigned to SUR1 subunits and consider whether these might be involved in the sequelae of ischemia/reperfusion
— id: 101292, year: 2009, vol: 19, page: 61, stat: Journal Article,

Calmodulin Kinase Inhibition Prevents the Arrhythmogenesis in Ryr2r4496c+/- Knock in Mice
Liu, NA; Ruan, YF; Denegri, M; Tiziana, B; Li, Y; Colombi, B; Napolitano, C; Coetzee, WA; Priori, SG
2009 NOV 3 ;120(18):S668-S668, Circulation
— id: 106977, year: 2009, vol: 120, page: S668, stat: Journal Article,

Role of sulfonylurea receptor type 1 subunits of ATP-sensitive potassium channels in myocardial ischemia/reperfusion injury
Elrod, John W; Harrell, Maddison; Flagg, Thomas P; Gundewar, Susheel; Magnuson, Mark A; Nichols, Colin G; Coetzee, William A; Lefer, David J
2008 Mar 18;117(11):1405-1413, Circulation
BACKGROUND: Opening of cardiac ATP-sensitive potassium channels (K(ATP) channels) is a well-characterized protective mechanism against ischemia and reperfusion injury. Evidence exists for an involvement of both sarcolemmal and mitochondrial K(ATP) channels in such protection. Classically, cardiac sarcolemmal K(ATP) channels are thought to be composed of Kir6.2 (inward-rectifier potassium channel 6.2) and SUR2A (sulfonylurea receptor type 2A) subunits; however, the evidence is strong that SUR1 (sulfonylurea receptor type 1) subunits are also expressed in the heart and that they may have a functional role. The aim of this study, therefore, was to examine the role of SUR1 in myocardial infarction. METHODS AND RESULTS: We subjected mice lacking SUR1 subunits to in vivo myocardial ischemia/reperfusion injury. Interestingly, the SUR1-null mice were markedly protected against the ischemic insult, displaying a reduced infarct size and preservation of left ventricular function, which suggests a role for this K(ATP) channel subunit in cardiovascular function during conditions of stress. CONCLUSIONS: SUR1 subunits have a high sensitivity toward many sulfonylureas and certain K(ATP) channel-opening drugs. Their potential role during ischemic events should therefore be considered both in the interpretation of experimental data with pharmacological agents and in the clinical arena when the cardiovascular outcome of patients treated with antidiabetic sulfonylureas is being considered
— id: 96217, year: 2008, vol: 117, page: 1405, stat: Journal Article,

Differential structure of atrial and ventricular KATP: atrial KATP channels require SUR1
Flagg, Thomas P; Kurata, Harley T; Masia, Ricard; Caputa, George; Magnuson, Mark A; Lefer, David J; Coetzee, William A; Nichols, Colin G
2008 Dec 5;103(12):1458-1465, Circulation research
The isoform-specific structure of the ATP-sensitive potassium (K(ATP)) channel endows it with differential fundamental properties, including physiological activation and pharmacology. Numerous studies have convincingly demonstrated that the pore-forming Kir6.2 (KCNJ11) and regulatory SUR2A (ABCC9) subunits are essential elements of the sarcolemmal K(ATP) channel in cardiac ventricular myocytes. Using a novel antibody directed against the COOH terminus of SUR1 (ABCC8), we show that this K(ATP) subunit is also expressed in mouse myocardium and is the dominant SUR isoform in the atrium. This suggests differential sarcolemmal K(ATP) composition in atria and ventricles, and, to test this, K(ATP) currents were measured in isolated atrial and ventricular myocytes from wild-type and SUR1(-/-) animals. K(ATP) conductance is essentially abolished in SUR1(-/-) atrial myocytes but is normal in SUR1(-/-) ventricular myocytes. Furthermore, pharmacological properties of wild-type atrial K(ATP) match closely the properties of heterologously expressed SUR1/Kir6.2 channels, whereas ventricular K(ATP) properties match those of heterologously expressed SUR2A/Kir6.2 channels. Collectively, the data demonstrate a previously unappreciated K(ATP) channel heterogeneity: SUR1 is an essential component of atrial, but not ventricular, K(ATP) channels. Differential molecular make-up of the 2 channels underlies differential pharmacology, with important implications when considering sulfonylurea therapy or dissecting the role of cardiac K(ATP) pharmacologically, as well as for understanding of the role of diazoxide in preconditioning
— id: 96216, year: 2008, vol: 103, page: 1458, stat: Journal Article,

Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic beta-cells and rat INS-1 cells
Kang, Guoxin; Leech, Colin A; Chepurny, Oleg G; Coetzee, William A; Holz, George G
2008 Mar 1;586(5):1307-1319, Journal of physiology
Protein kinase A (PKA)-independent actions of adenosine 3',5'-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic beta-cells. Evidence that Epac might mediate the cAMP-dependent inhibition of beta-cell ATP-sensitive K(+) channels (K(ATP)) was provided by one prior study of human beta-cells and a rat insulin-secreting cell line (INS-1 cells) in which it was demonstrated that an Epac-selective cAMP analogue (ESCA) inhibited a sulphonylurea-sensitive K(+) current measured under conditions of whole-cell recording. Using excised patches of plasma membrane derived from human beta-cells and rat INS-1 cells, we now report that 2'-O-Me-cAMP, an ESCA that activates Epac but not PKA, sensitizes single K(ATP) channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2'-O-Me-cAMP (50 microM), the dose-response relationship describing ATP-dependent inhibition of K(ATP) channel activity (NP(o)) is left-shifted such that the concentration of ATP producing 50% inhibition (IC(50)) is reduced from 22 microM to 1 microM for human beta-cells, and from 14 microM to 4 microM for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 microM), the administration of 2'-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC(50) 12 microM for both cell types). A cyclic nucleotide phosphodiesterase-resistant ESCA (Sp-8-pCPT-2'-O-Me-cAMPS) also inhibits K(ATP) channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human beta-cells and rat INS-1 cells a novel form of ion channel modulation in which the ATP sensitivity of K(ATP) channels is regulated by Epac
— id: 79136, year: 2008, vol: 586, page: 1307, stat: Journal Article,

Decreased connexin43 expression in the mouse heart potentiates pacing-induced remodeling of repolarizing currents
Kontogeorgis, Andrianos; Li, Xiaodong; Kang, Eunice Y; Feig, Jonathan E; Ponzio, Marc; Kang, Guoxin; Kaba, Riyaz A; Wit, Andrew L; Fisher, Edward A; Morley, Gregory E; Peters, Nicholas S; Coetzee, William A; Gutstein, David E
2008 Nov;295(5):H1905-H1916, American journal of physiology. Heart & circulatory physiology
Gap junction redistribution and reduced expression, a phenomenon termed gap junction remodeling (GJR), is often seen in diseased hearts and may predispose towards arrhythmias. We have recently shown that short-term pacing in the mouse is associated with changes in connexin43 (Cx43) expression and localization, but not with increased inducibility into sustained arrhythmias. We hypothesized that short-term pacing, if imposed on murine hearts with decreased Cx43 abundance, could serve as a model for evaluating the electrophysiologic effects of GJR. We paced wildtype (normal Cx43 abundance) and heterozygous Cx43 knockout mice (Cx43(+/-), 66% mean reduction in Cx43) for six hours at 10-15% above their average sinus rate. We investigated the electrophysiologic effects of pacing on the whole animal using programmed electrical stimulation, and in isolated ventricular myocytes with patch clamp studies. Cx43(+/-) myocytes had significantly shorter action potential durations (APD) and increased steady state and inward rectifier potassium currents (Iss and IK1, respectively) compared to wildtype littermate cells. In Cx43(+/-) hearts, pacing resulted in significant prolongation of ventricular effective refractory period and action potential duration, and significant diminution of Iss compared to unpaced Cx43(+/-) hearts. However, these changes were not seen in paced wildtype mice. These data suggest that Cx43 abundance plays a critical role in regulating currents involved in myocardial repolarization and their response to pacing. Our study may aid in understanding how dyssynchronous activation of diseased, Cx43-deficient myocardial tissue can lead to electrophysiologic changes which may contribute to the worsened prognosis often associated with pacing in the failing heart. Key words: Connexin43, ventricular myocytes, mouse, gap junction
— id: 116200, year: 2008, vol: 295, page: H1905, stat: Journal Article,

Functional and pharmacological characterization of a Shal-related K+ channel subunit in Zebrafish
Nakamura, Tomoe Y; Coetzee, William A
2008 ;8:2-2, BMC Physiology
BACKGROUND: K+ channels are diverse; both in terms of their function and their molecular composition. Shal subunits were first described in Drosophila. There are three mammalian orthologs, which are members of the Kv4 subfamily. They are involved in neuronal firing patterns as well as control of the cardiac action potential duration. RESULTS: Here, we report the biophysical and pharmacological characterization of zShal3, which is the ortholog of the mammalian Kv4.3 subunit, which in mammals is involved in action potential repolarization and gives rise to neuronal A-type K+ currents involved in somatodendretic signal integration. CONCLUSION: We demonstrate that zShal has similar functional and pharmacological characteristics compared to Kv4.3 and it is similarly regulated by pharmacological agents and by the Kv4 accessory subunit, NCS-1
— id: 79131, year: 2008, vol: 8, page: 2, stat: Journal Article,

Expression of a sorcin missense mutation in the heart modulates excitation-contraction coupling
Collis, Leon P; Meyers, Marian B; Zhang, Jie; Phoon, Colin K L; Sobie, Eric A; Coetzee, William A; Fishman, Glenn I
2007 Feb;21(2):475-487, FASEB journal
Sorcin is a Ca2+ binding protein implicated in the regulation of intracellular Ca2+ cycling and cardiac excitation-contraction coupling. Structural and human genetic studies suggest that a naturally occurring sequence variant encoding L112-sorcin disrupts an E-F hand Ca2+ binding domain and may be responsible for a heritable form of hypertension and hypertrophic heart disease. We generated transgenic mice overexpressing L112-sorcin in the heart and characterized the effects on Ca2+ regulation and cardiac function both in vivo and in dissociated cardiomyocytes. Hearts of sorcin(F112L) transgenic mice were mildly dilated but ventricular function was preserved and systemic blood pressure was normal. Sorcin(F112L) myocytes were smaller than control cells and displayed complex alterations in Ca2+ regulation and contractility, including a slowed inactivation of L-type Ca2+ current, enhanced Ca2+ spark width, duration, and frequency, and increased Na+-Ca2+ exchange activity. In contrast, mice with cardiac-specific overexpression of wild-type sorcin displayed directionally opposite effects on L-type Ca2+ channel function and Ca2+ spark behavior. These data further define the role of sorcin in cardiac excitation-contraction coupling and highlight its negative regulation of SR calcium release. Our results also suggest that additional factors may be responsible for the development of cardiac hypertrophy and hypertension in humans expressing the L112-sorcin sequence variant.
— id: 72805, year: 2007, vol: 21, page: 475, stat: Journal Article,

beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes
Collis, Leon P; Srivastava, Shekhar; Coetzee, William A; Artman, Michael
2007 Nov;293(5):H2826-H2835, American journal of physiology. Heart & circulatory physiology
Selective stimulation of beta(2)-adrenergic receptors (ARs) in newborn rabbit ventricular myocardium invokes a positive inotropic effect that is lost during postnatal maturation. The underlying mechanisms for this age-related stimulatory response remain unresolved. We examined the effects of beta(2)-AR stimulation on L-type Ca(2+) current (I(Ca,L)) during postnatal development. I(Ca,L) was measured (37 degrees C; either Ca(2+) or Ba(2+) as the charge carrier) using the whole-cell patch-clamp technique in newborn (1 to 5 days old) and adult rabbit ventricular myocytes. Ca(2+) transients were measured concomitantly by dialyzing the cell with indo-1. Activation of beta(2)-ARs (with either 100 nM zinterol or 1 microM isoproterenol in the presence of the beta(1)-AR antagonist, CGP20712A) stimulated I(Ca,L) twofold in newborns but not in adults. The beta(2)-AR-mediated increase in Ca(2+) transient amplitude in newborns was due exclusively to the augmentation of I(Ca,L). Zinterol increased the rate of inactivation of I(Ca,L) and increased the Ca(2+) flux integral. The beta(2)-AR inverse agonist, ICI-118551 (500 nM), but not the beta(1)-AR antagonist, CGP20712A (500 nM), blocked the response to zinterol. Unexpectedly, the PKA blockers, H-89 (10 microM), PKI 6-22 amide (10 microM), and Rp-cAMP (100 microM), all failed to prevent the response to zinterol but completely blocked responses to selective beta(1)-AR stimulation of I(Ca,L) in newborns. Our results demonstrate that in addition to the conventional beta(1)-AR/cAMP/PKA pathway, newborn rabbit myocardium exhibits a novel beta(2)-AR-mediated, PKA-insensitive pathway that stimulates I(Ca,L). This striking developmental difference plays a major role in the age-related differences in inotropic responses to beta(2)-AR agonists
— id: 75449, year: 2007, vol: 293, page: H2826, stat: Journal Article,

The regulation of ion channels and transporters by glycolytically derived ATP
Dhar-Chowdhury, P; Malester, B; Rajacic, P; Coetzee, W A
2007 Dec;64(23):3069-3083, Cellular & molecular life sciences: CMLS
Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na(+)/K(+)-ATPase, the H(+)-ATPase, various types of Ca(2+)-ATPases, the Na(+)/H(+) exchanger, the ATP-sensitive K(+) channel, cation channels, Na(+) channels, Ca(2+) channels and other channels involved in intracellular Ca(2+) homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches
— id: 75763, year: 2007, vol: 64, page: 3069, stat: Journal Article,

Differential make-up of atrial and ventricular KA
Flagg, TP; Kurata, HT; Coetzee, WA; Lefer, DJ; Magnuson, MA; Nichols, CG
2007 NOV 26 ;101(11):1211-1211, Circulation research
— id: 75214, year: 2007, vol: 101, page: 1211, stat: Journal Article,

Large-scale analysis of ion channel gene expression in the mouse heart during perinatal development
Harrell, M D; Harbi, S; Hoffman, J F; Zavadil, J; Coetzee, W A
2007 Feb 12;28(3):273-283, Physiological genomics
The immature and mature heart differ from each other in terms of excitability, action potential properties, contractility, and relaxation. This includes upregulation of repolarizing K(+) currents, an enhanced inward rectifier K(+) (Kir) current, and changes in Ca(2+), Na(+), and Cl(-) currents. At the molecular level, the developmental regulation of ion channels is scantily described. Using a large-scale real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, we performed a comprehensive analysis of ion channel transcript expression during perinatal development in the embryonic (embryonic day 17.5), neonatal (postnatal days 1-2), and adult Swiss-Webster mouse hearts. These data are compared with publicly available microarray data sets (Cardiogenomics project). Developmental mRNA expression for several transcripts was consistent with the published literature. For example, transcripts such as Kir2.1, Kir3.1, Nav1.5, Cav1.2, etc. were upregulated after birth, whereas others [e.g., Ca(2+)-activated K(+) (KCa)2.3 and minK] were downregulated. Cl(-) channel transcripts were expressed at higher levels in immature heart, particularly those that are activated by intracellular Ca(2+). Defining alterations in the ion channel transcriptome during perinatal development will lead to a much improved understanding of the electrophysiological alterations occurring in the heart after birth. Our study may have important repercussions in understanding the mechanisms and consequences of electrophysiological alterations in infants and may pave the way for better understanding of clinically relevant events such as congenital abnormalities, cardiomyopathies, heart failure, arrhythmias, cardiac drug therapy, and the sudden infant death syndrome
— id: 71580, year: 2007, vol: 28, page: 273, stat: Journal Article,

Modulation of human cardiovascular outward rectifying chloride channel by intra- and extracellular ATP
Liu, Gong Xin; Vepa, Sanjay; Artman, Michael; Coetzee, William A
2007 Dec;293(6):H3471-H3479, American journal of physiology. Heart & circulatory physiology
The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl(-) channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out single-channel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl(-) solutions, exhibited a slope conductance of approximately 90-100 pS at positive potentials and approximately 22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, 'extracellular' ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATPgammaS > ATP = UTP > ADP > AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions
— id: 93831, year: 2007, vol: 293, page: H3471, stat: Journal Article,

Transgenic expression of a dominant negative K(ATP) channel subunit in the mouse endothelium: effects on coronary flow and endothelin-1 secretion
Malester, Brian; Tong, Xiaoyong; Ghiu, Ioana; Kontogeorgis, Andrianos; Gutstein, David E; Xu, Jie; Hendricks-Munoz, Karen D; Coetzee, William A
2007 Jul;21(9):2162-2172, FASEB journal
K(ATP) channels are involved in regulating coronary function, but the contribution of endothelial K(ATP) channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial K(ATP) channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothelium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine-induced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The K(ATP) channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the K(ATP) channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin-1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin-1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin-1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of K(ATP) channels led to elevated endothelin-1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin-1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial K(ATP) channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothelin-1
— id: 73407, year: 2007, vol: 21, page: 2162, stat: Journal Article,

Histidine phosphorylation of the Ca2+-activated K+ channel KCa3.1 by nucleoside diphosphate kinase B (NDPK-B) is required for KCa3.1 channel activation and the reactivation of CD4 T lymphocytes
Srivastava, S; Li, Z; Ko, K; Choudhury, P; Albaqumi, M; Johnson, AK; Yan, Y; Backer, J; Unutmaz, D; Coetzee, WA; Skolnik, EY
2007 JAN ;108(11):200A-200A, Biophysical journal
— id: 71388, year: 2007, vol: 108, page: 200A, stat: Journal Article,

Phospholemman expression is high in the newborn rabbit heart and declines with postnatal maturation
Srivastava, Shekhar; Cala, Steven E; Coetzee, William A; Artman, Michael
2007 Apr 6;355(2):338-341, Biochemical & biophysical research communications
Phospholemman (PLM) is a small sarcolemmal protein that modulates the activities of Na(+)/K(+)-ATPase and the Na(+)/Ca(2+) exchanger (NCX), thus contributing to the maintenance of intracellular Na(+) and Ca(2+) homeostasis. We characterized the expression and subcellular localization of PLM, NCX, and the Na(+)/K(+)-ATPase alpha1-subunit during perinatal development. Western blotting demonstrates that PLM (15kDa), NCX (120kDa), and Na(+)/K(+)-ATPase alpha-1 (approximately 100kDa) proteins are all more than 2-fold higher in ventricular membrane fractions from newborn rabbit hearts (1-4-day old) compared to adult hearts. Our immunocytochemistry data demonstrate that PLM, NCX, and Na(+)/K(+)-ATPase are all expressed at the sarcolemma of newborn ventricular myocytes. Taken together, our data indicate that PLM, NCX, and Na(+)/K(+)-ATPase alpha-1 proteins have similar developmental expression patterns in rabbit ventricular myocardium. Thus, PLM may have an important regulatory role in maintaining cardiac Na(+) and Ca(2+) homeostasis during perinatal maturation
— id: 71420, year: 2007, vol: 355, page: 338, stat: Journal Article,

Specificity of the myotubularin family of phosphatidylinositol-3-phosphatase is determined by the PH/GRAM domain
Choudhury, Papiya; Srivastava, Shekhar; Li, Zhai; Ko, Kyung; Albaqumi, Mamdouh; Narayan, Kartik; Coetzee, William A; Lemmon, Mark A; Skolnik, Edward Y
2006 Oct 20;281(42):31762-31769, Journal of biological chemistry
Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease
— id: 70220, year: 2006, vol: 281, page: 31762, stat: Journal Article,

SUR1 knockout mice are protected against myocardial ischemia-reperfusion injury
Elrod, JW; Malester, B; Harrell, MD; Duranski, MR; Magnuson, MA; Lefer, DJ; Coetzee, WA
2006 OCT 31 ;114(18):274-274, Circulation
— id: 69548, year: 2006, vol: 114, page: 274, stat: Journal Article,

cAMP Sensor Epac As A Determinant Of ATP-Sensitive Potassium Channel Activity In Human Pancreatic Beta Cells And Rat INS-1 Cells
Kang, Guoxin; Chepurny, Oleg G; Malester, Brian; Rindler, Michael J; Rehmann, Holger; Bos, Johannes L; Schwede, Frank; Coetzee, William A; Holz, George G
2006 Jun 15;573(Pt 3):595-609, Journal of physiology
The Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic beta cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide-binding fold-1 (NBF-1) of the beta cell sulfonylurea receptor-1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit beta cell ATP-sensitive K+ channels (KATP channels; a hetero-octomer of SUR1 and Kir6.2). If so, Epac-mediated inhibition of KATP channels might explain prior reports that cAMP-elevating agents promote beta cell depolarization, Ca2+ influx, and insulin secretion. Here we report that Epac-selective cAMP analogs (2'-O-Me- cAMP; 8-pCPT-2'-O-Me-cAMP; 8-pMeOPT-2'-O-Me-cAMP), but not a cGMP analog (2'-O-Me-cGMP), inhibit the function of KATP channels in human beta cells and rat INS-1 insulin-secreting cells. Inhibition of KATP channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6-Bnz-cAMP, a cAMP analog that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac-selective cAMP analogs at KATP channels are mimicked by a cAMP agonist (Sp-8-Br-cAMPS), but not a cAMP antagonist (Rp-8-Br-cAMPS), and are abrogated following transfection of INS-1 cells with a dominant- negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 co-immunoprecipitate with full-length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of KATP channel activity in pancreatic beta cells
— id: 63619, year: 2006, vol: 573, page: 595, stat: Journal Article,

Phosphatidylinositol 3-Phosphate Indirectly Activates KCa3.1 via 14 Amino Acids in the Carboxy Terminus of KCa3.1
Srivastava, Shekhar; Choudhury, Papiya; Li, Zhai; Liu, Gongxin; Nadkarni, Vivek; Ko, Kyung; Coetzee, William A; Skolnik, Edward Y
2006 Jan;17(1):146-154, Molecular biology of the cell
Monitoring Editor: Guido Guidotti KCa3.1 is an intermediate conductance Ca(2+)-activated K channels that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume and chloride secretion. We recently found that the KCa3.1 channel also specifically requires PI(3)P for channel activity and is inhibited by myotubularin related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2 or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for their biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxyterminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data when taken together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1
— id: 59246, year: 2006, vol: 17, page: 146, stat: Journal Article,

Phosphatidylinositol-3 phosphatase myotubularin-related protein 6 negatively regulates CD4 T cells
Srivastava, Shekhar; Ko, Kyung; Choudhury, Papiya; Li, Zhai; Johnson, Amanda K; Nadkarni, Vivek; Unutmaz, Derya; Coetzee, William A; Skolnik, Edward Y
2006 Aug;26(15):5595-5602, Molecular & cellular biology
Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell
— id: 68660, year: 2006, vol: 26, page: 5595, stat: Journal Article,

Histidine phosphorylation of the potassium channel KCa3.1 by nucleoside diphosphate kinase B is required for activation of KCa3.1 and CD4 T cells
Srivastava, Shekhar; Li, Zhai; Ko, Kyung; Choudhury, Papiya; Albaqumi, Mamdouh; Johnson, Amanda K; Yan, Ying; Backer, Jonathan M; Unutmaz, Derya; Coetzee, William A; Skolnik, Edward Y
2006 Dec 8;24(5):665-675, Molecular cell
The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation
— id: 69707, year: 2006, vol: 24, page: 665, stat: Journal Article,

Consequences of Cardiac Myocyte-Specific Ablation of KATP channels in Transgenic Mice expressing Dominant Negative Kir6 Subunits
Tong, XiaoYong; Porter, Lisa M; Liu, GongXin; Dhar-Chowdhury, Piyali; Srivastava, Shekhar; Pountney, David J; Yoshida, Hidetada; Artman, Michael; Fishman, Glenn I; Yu, Cindy; Iyer, Ramesh; Morley, Gregory E; Gutstein, David E; Coetzee, William A
2006 Aug;291(2):H543-H551, American journal of physiology. Heart & circulatory physiology
Cardiac KATP channels are formed by Kir6.2 and SUR2A subunits. We produced transgenic mice which express dominant negative Kir6.x pore-forming subunits (Kir6.1-AAA or Kir6.2-AAA) in cardiac myocytes by driving their expression with the alpha-myosin heavy chain promoter. Weight gain and development after birth of these mice were similar to wild-type mice, but an increased mortality was noted after the age of 4-5 months. Transgenic mice lacked cardiac KATP channel activity as assessed with patch clamp techniques. Consistent with a decreased current density observed at positive voltages, the action potential duration was increased in these mice. Some myocytes developed early afterdepolarizations following isoproterenol treatment. Hemodynamic measurements revealed no significant effects on ventricular function (apart from a slightly elevated heart rate) whereas in-vivo electrophysiological recordings revealed a prolonged ventricular effective refractory period in transgenic mice. The transgenic mice tolerated stress less well as evident from treadmill stress tests. The pro-arrhythmogenic features and lack of adaptation to a stress response in transgenic mice suggests that these features are intrinsic to the myocardium and that KATP channels in the myocardium have an important role in protecting the heart from lethal arrhythmias and adaptation to stress situations
— id: 63616, year: 2006, vol: 291, page: H543, stat: Journal Article,

The Glycolytic Enzymes, Glyceraldehyde-3-phosphate Dehydrogenase, Triose-phosphate Isomerase, and Pyruvate Kinase Are Components of the KATP Channel Macromolecular Complex and Regulate Its Function
Dhar-Chowdhury, Piyali; Harrell, Maddison D; Han, Sandra Y; Jankowska, Danuta; Parachuru, Lavanya; Morrissey, Alison; Srivastava, Shekhar; Liu, Weixia; Malester, Brian; Yoshida, Hidetada; Coetzee, William A
2005 Nov 18;280(46):38464-38470, Journal of biological chemistry
The regulation of ATP-sensitive potassium (K(ATP)) channel activity is complex and a multitude of factors determine their open probability. Physiologically and pathophysiologically, the most important of these are intracellular nucleotides, with a long-recognized role for glycolytically derived ATP in regulating channel activity. To identify novel regulatory subunits of the K(ATP) channel complex, we performed a two-hybrid protein-protein interaction screen, using as bait the mouse Kir6.2 C terminus. Screening a rat heart cDNA library, we identified two potential interacting proteins to be the glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triose-phosphate isomerase. The veracity of interaction was verified by co-immunoprecipitation techniques in transfected mammalian cells. We additionally demonstrated that pyruvate kinase also interacts with Kir6.2 subunits. The physiological relevance of these interactions is illustrated by the demonstration that native Kir6.2 protein similarly interact with GAPDH and pyruvate kinase in rat heart membrane fractions and that Kir6.2 protein co-localize with these glycolytic enzymes in rat ventricular myocytes. The functional relevance of our findings is demonstrated by the ability of GAPDH or pyruvate kinase substrates to directly block the K(ATP) channel under patch clamp recording conditions. Taken together, our data provide direct evidence for the concept that key enzymes involved in glycolytic ATP production are part of a multisubunit K(ATP) channel protein complex. Our data are consistent with the concept that the activity of these enzymes (possibly by ATP formation in the immediate intracellular microenvironment of this macromolecular K(ATP) channel complex) causes channel closure
— id: 59247, year: 2005, vol: 280, page: 38464, stat: Journal Article,

Negative inotropic effect of nifedipine in the immature rabbit heart is due to shortening of the action potential
Go, Anita; Srivastava, Shekhar; Collis, Leon; Coetzee, William A; Artman, Michael
2005 Mar;57(3):399-403, Pediatric research
Contractions in neonatal rabbit ventricular myocytes seem to depend predominantly on Ca2+ influx through the Na+-Ca2+ exchanger (NCX). Unexpectedly, neonates are sensitive to the negative inotropic effect of L-type Ca2+ channel blockers. L-type Ca2+ channel blockers depress contractile function indirectly in neonatal myocytes by shortening the action potential duration (APD), thereby decreasing the influx of activator Ca2+ through the NCX. Freshly isolated ventricular myocytes from adult and neonatal (1-5 d) rabbits were electrically stimulated (0.5 Hz; 35 degrees C) while action potential (AP) and Ca2+ transients (Indo-1) were recorded in the absence and presence of nifedipine (10 microM). In separate experiments, cells were voltage-clamped with a constant AP waveform (APD90=170 ms) to determine the effect of nifedipine on Ca2+ transients independent from effects on the AP. Voltage-clamp experiments confirmed that nifedipine blocks L-type Ca2+ current in neonatal myocytes. Nifedipine markedly reduced Ca2+ transient amplitude and APD in both adults (transient=20 +/- 7%; APD90=31 +/- 4% of control) and neonates (transient=38 +/- 10%; APD90=57 +/- 6% of control). When the AP was held constant by voltage clamping, nifedipine significantly reduced the amplitude of Ca2+ transients in adults (27 +/- 9% of control) but had no effect on Ca2+ transient amplitude in neonatal myocytes. These results are consistent with the concept that immature ventricular myocytes are less reliant on L-type calcium channels and are more dependent on NCX for contraction. The negative inotropic effect of L-type Ca2+ channel blockers in neonates is attributable to shortening of the AP
— id: 51753, year: 2005, vol: 57, page: 399, stat: Journal Article,

Expression of ATP-sensitive K+ channel subunits during perinatal maturation in the mouse heart
Morrissey, Alison; Parachuru, Lavanya; Leung, Monika; Lopez, Gwendolyn; Nakamura, Tomoe Y; Tong, Xiaoyong; Yoshida, Hidetada; Srivastiva, Shekhar; Chowdhury, Piyali Dhar; Artman, Michael; Coetzee, William A
2005 Aug;58(2):185-192, Pediatric research
Prevailing data suggest that sarcolemmal ATP-sensitive (K(ATP)) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other K(ATP) channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal K(ATP) channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of K(ATP) channels of immature and adult K(ATP) channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various K(ATP) channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription-PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of K(ATP) channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the K(ATP) channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the K(ATP) channel composition may change during maturation, which has important implications for K(ATP) channel function in the developing heart
— id: 58895, year: 2005, vol: 58, page: 185, stat: Journal Article,

Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature
Morrissey, Alison; Rosner, Erika; Lanning, Jennifer; Parachuru, Lavanya; Dhar Chowdhury, Piyali; Han, Sandra; Lopez, Gwendolyn; Tong, XiaoYong; Yoshida, Hidetada; Nakamura, Tomoe Y; Artman, Michael; Giblin, Jonathan P; Tinker, Andrew; Coetzee, William A
2005 Jan 12;5(1):1-1, BMC Physiology
BACKGROUND: Electrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart. RESULTS: Immunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels. CONCLUSIONS: Collectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures
— id: 55958, year: 2005, vol: 5, page: 1, stat: Journal Article,

Paradoxical effect of dofetilide on action potential duration and calcium transient amplitude in newborn rabbit ventricular myocytes
Srivastava, Shekhar; Collis, Leon; Go, Anita; Mancarella, Salvatore; Coetzee, William A; Artman, Michael
2005 Feb;45(2):165-174, Journal of cardiovascular pharmacology
The Na+-Ca2+ exchanger (NCX) is up-regulated in the neonatal rabbit heart. Because the duration of membrane depolarization is an important determinant of calcium entry via NCX, pharmacological agents that lengthen the action potential (AP) may significantly increase the amount of activator calcium in newborns. We tested this potentially novel therapeutic strategy by using action potential voltage clamp steps or using dofetilide, a blocker of IKr, to prolong the action potential duration (APD). The effects of changing APD on calcium transients were determined in ventricular myocytes at different developmental stages: newborn (1-4 days), juvenile (9-10 days), and adult ventricular myocytes (35 degrees C; 1 Hz). Calcium transient amplitude in neonatal myocytes increased substantially with clamping with longer APs. In contrast, exposure to dofetilide (0.1, 1, and 10 microM) under current clamp conditions increased APD in a concentration-dependent manner but had no significant effect on calcium transient amplitude in either neonates or adults. When the AP was held constant under voltage clamp conditions, dofetilide decreased the calcium transient amplitude in neonates. This effect is likely related to inhibition of sodium-calcium exchanger and L-type Ca2+ currents (ICa), as observed in separate experiments. These results suggest that dofetilide has a paradoxical effect on APD and calcium transients in the newborn heart
— id: 51784, year: 2005, vol: 45, page: 165, stat: Journal Article,

The phosphatidylinositol 3-phosphate phosphatase myotubularin- related protein 6 (MTMR6) is a negative regulator of the Ca2+-activated K+ channel KCa3.1
Srivastava, Shekhar; Li, Zhai; Lin, Lin; Liu, GongXin; Ko, Kyung; Coetzee, William A; Skolnik, Edward Y
2005 May;25(9):3630-3638, Molecular & cellular biology
Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3' position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P(2). MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca(2+)-activated K channel, K(Ca)3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K(Ca)3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K(Ca)3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K(Ca)3.1, indicating that MTM CC domains are sufficient to confer target specificity. K(Ca)3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K(Ca)3.1 by MTMR6 overexpression. These data, when taken together, indicate that K(Ca)3.1 is regulated by PI(3)P and that MTMR6 inhibits K(Ca)3.1 by dephosphorylating the 3' position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K(Ca)3.1. K(Ca)3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K(Ca)3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis
— id: 55912, year: 2005, vol: 25, page: 3630, stat: Journal Article,

K-ATP channels regulate ET-1 release in human coronary arterial endothelial cells
Ghiu, IA; Yoshida, H; Feig, J; Morrissey, A; Coetzee, WA
2004 JAN ;86(1):441A-441A, Biophysical journal
— id: 42459, year: 2004, vol: 86, page: 441A, stat: Journal Article,

Myotubularins (MTMs) are lipid phosphatases that negatively regulate the Ca2+-activated K+ channel (KCa3.1)
Lin, L; Li, Z; Coetzee, WA; Skolnik, EY
2004 OCT 26 ;110(17):62-62, Circulation
— id: 55935, year: 2004, vol: 110, page: 62, stat: Journal Article,

Developmental differences in the effects of beta-adrenergic receptor agonists on calcium transients and sodium-calcium exchange currents
Mancarella, S; Coetzee, W; Artman, M
2004 JAN ;86(1):555A-555A, Biophysical journal
— id: 42462, year: 2004, vol: 86, page: 555A, stat: Journal Article,

Developmental expression of phospholemman in rabbit and mouse heart
Srivastava, S; Coetzee, WA; Cala, SE; Artman, M
2004 JAN ;86(1):262A-262A, Biophysical journal
— id: 42457, year: 2004, vol: 86, page: 262A, stat: Journal Article,

Native K-ATP channels in human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits
Yoshida, H; Feig, JE; Coetzee, WA
2004 JAN ;86(1):440A-441A, Biophysical journal
— id: 42458, year: 2004, vol: 86, page: 440A, stat: Journal Article,

K ATP channels of primary human coronary artery endothelial cells consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits
Yoshida, Hidetada; Feig, Jonathan E; Morrissey, Alison; Ghiu, Ioana A; Artman, Michael; Coetzee, William A
2004 Oct;37(4):857-869, Journal of molecular & cellular cardiology
Functional ATP-sensitive potassium (K(ATP)) channels can be reconstituted by expression of various combinations of different pore-forming subunits (Kir6.1 and Kir6.2) and sulfonylurea receptor (SUR) subunits. Using dominant negative and gene knockout approaches, Kir6.2 subunits have been identified as required pore-forming components of plasmalemmal K(ATP) channels in ventricular myocytes. Previous data obtained in heterologous expression systems suggest that Kir6.1 and Kir6.2 subunits are capable of forming a functional heteromultimeric channel complex. However, until now the existence of such heteromultimeric Kir6.1/Kir6.2 complexes has not been demonstrated for native K(ATP) channels. The primary aim of this study was to identify the molecular composition of native K(ATP) channels in primary human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) from human origin. We specifically investigated the potential that heteromultimeric Kir6.1/Kir6.2 channels exist in these cells. Using reverse transcriptase-polymerase chain reaction, we detected the expression of Kir6.1, Kir6.2, and SUR2B in both cell types. Western blotting and immunoprecipitation assays demonstrated the presence of Kir6.1 protein in both HCAEC and HCASMC; however, Kir6.2 protein was only expressed in HCAEC. Interaction between Kir6.1 and Kir6.2 subunits was demonstrated by reciprocal co-immunoprecipitation of these two subunits in HCAEC. Furthermore, Kir6.1 and Kir6.2 were detected in the immunoprecipitate when using an anti-SUR2 antibody. Confocal microscopy imaging demonstrated Kir6.1 and Kir6.2 subunits to co-localize at the cell surface membrane in HCAEC. In conclusion, our data characterize the molecular composition of primary human coronary smooth muscle and endothelial cells. We demonstrate that human coronary endothelial K(ATP) channels consist of a heteromultimeric complex of Kir6.1, Kir6.2, and SUR2B subunits
— id: 48872, year: 2004, vol: 37, page: 857, stat: Journal Article,

A molecular basis for the sensitivity of cardiac K-ATP channels to glycolytic ATP
Chowdhury, PD; Jankowska, D; Liu, WX; Han, S; Coetzee, WA
2003 OCT 28 ;108(17):211-212, Circulation
— id: 42526, year: 2003, vol: 108, page: 211, stat: Journal Article,

Maintaining a constant action potential prevents the negative inotropic effect of nifedipine in neonatal ventricular myocytes
Go, A; Srivastava, S; Coetzee, WA; Artman, M
2003 APR ;53(4):35A-35A, Pediatric research
— id: 38558, year: 2003, vol: 53, page: 35A, stat: Journal Article,

beta(2)-adrenergic stimulation increases Na+/Ca2+ exchanger current in neonatal rabbit ventricular myocytes
Mancarella, S; Collis, L; Coetzee, W; Artman, M
2003 JUN ;35(6):A31-A31, Journal of molecular & cellular cardiology
— id: 38487, year: 2003, vol: 35, page: A31, stat: Journal Article,

Sorcin regulates excitation-contraction coupling in the heart
Meyers, Marian B; Fischer, Avi; Sun, Yan-Jie; Lopes, Coeli M B; Rohacs, Tibor; Nakamura, Tomoe Y; Zhou, Ying-Ying; Lee, Paul C; Altschuld, Ruth A; McCune, Sylvia A; Coetzee, William A; Fishman, Glenn I
2003 Aug 1;278(31):28865-28871, Journal of biological chemistry
Sorcin is a penta-EF hand Ca2+-binding protein that associates with both cardiac ryanodine receptors and L-type Ca2+ channels and has been implicated in the regulation of intracellular Ca2+ cycling. To better define the function of sorcin, we characterized transgenic mice in which sorcin was overexpressed in the heart. Transgenic mice developed normally with no evidence of cardiac hypertrophy and no change in expression of other calcium regulatory proteins. In vivo hemodynamics revealed significant reductions in global indices of contraction and relaxation. Contractile abnormalities were also observed in isolated adult transgenic myocytes, along with significant depression of Ca2+ transient amplitudes. Whole cell ICa density and the time course of activation were normal in transgenic myocytes, but the rate of inactivation was significantly accelerated. These effects of sorcin on L-type Ca2+ currents were confirmed in Xenopus oocyte expression studies. Finally, we examined the expression of sorcin in normal and failing hearts from spontaneous hypertensive heart failure rats. In normal myocardium, sorcin extensively co-localized with ryanodine receptors at the Z-lines, whereas in myopathic hearts the degree of co-localization was markedly disrupted. Together, these data indicate that sorcin modulates intracellular Ca2+ cycling and Ca2+ influx pathways in the heart
— id: 39224, year: 2003, vol: 278, page: 28865, stat: Journal Article,

Expression of K-ATP channel subunits in the human and murine heart
Morrissey, A; Rosner, E; Lanning, J; Lopez, G; Nakamura, TY; Ghiu, IA; Feig, J; Yoshida, H; Coetzee, WA
2003 FEB ;84(2):225A-225A, Biophysical journal
— id: 38574, year: 2003, vol: 84, page: 225A, stat: Journal Article,

Developmental expression of NCS-1 (frequenin), a regulator of Kv4 K+ channels, in mouse heart
Nakamura, Tomoe Y; Sturm, Eron; Pountney, David J; Orenzoff, Barbara; Artman, Michael; Coetzee, William A
2003 Apr;53(4):554-557, Pediatric research
The channel proteins responsible for the cardiac transient outward K+ current (Ito) of human and rodent heart are composed, in part, of pore-forming Kv4.3 or Kv4.2 principal subunits. Recent reports implicate K+ channel interacting proteins (members of the neuronal Ca2+-binding protein family) as subunits of the Ito channel complex. We reported that another Ca2+-binding protein, frequenin [or neuronal calcium center protein-1 (NCS-1)], also functions as a Kv4 auxiliary subunit in the brain. By examining cardiac expression of NCS-1, the aim of this study was to examine the potential physiologic relevance of this protein as an additional regulator of cardiac Ito. Immunoblot analysis demonstrates NCS-1 protein to be expressed in adult mouse ventricle at levels comparable to that found in some brain regions. Cardiac NCS-1 protein expression levels are much higher in fetal and neonatal mouse hearts when compared with the adult. Immunocytochemical analysis of isolated neonatal mouse ventricular myocytes demonstrates co-localization of NCS-1 and Kv4.2 proteins at the sarcolemma. Given its high levels of expression in the heart, NCS-1 should be considered an important potential Kv4 regulatory subunit, particularly in the immature heart
— id: 39285, year: 2003, vol: 53, page: 554, stat: Journal Article,

Evaluation of a potential newborn-specific approach to positive inotropic therapy
Srivastava, S; Holmes, D; Go, A; Coetzee, WA; Artman, M
2003 FEB ;84(2):258A-259A, Biophysical journal
— id: 38575, year: 2003, vol: 84, page: 258A, stat: Journal Article,

Identification and molecular characterization of native K-ATP channels in human coronary artery smooth muscle
Yoshida, H; Feig, JE; Morrissey, A; Ghiu, I; Artman, M; Coetzee, WA
2003 JUN ;35(6):A23-A23, Journal of molecular & cellular cardiology
— id: 38486, year: 2003, vol: 35, page: A23, stat: Journal Article,

5 '-adenosine monophosphate-activated protein kinase activates cardiac K-ATP channels
Yoshida, H; Kaneko, M; Coetzee, WA
2003 OCT 28 ;108(17):211-211, Circulation
— id: 42525, year: 2003, vol: 108, page: 211, stat: Journal Article,

Alpha1-adrenoceptor-mediated breakdown of phosphatidylinositol 4,5-bisphosphate inhibits pinacidil-activated ATP-sensitive K+ currents in rat ventricular myocytes
Haruna, Tetsuya; Yoshida, Hidetada; Nakamura, Tomoe Y; Xie, Lai-Hua; Otani, Hideo; Ninomiya, Tomonori; Takano, Makoto; Coetzee, William A; Horie, Minoru
2002 Aug 9;91(3):232-239, Circulation research
Phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates ATP-sensitive K+ (K(ATP)) channel activity. Because phospholipase C (PLC) hydrolyzes membrane-bound PIP2, which in turn may potentially decrease K(ATP) channel activity, we investigated the effects of the alpha1-adrenoceptor-G(q)-PLC signal transduction axis on pinacidil-activated K(ATP) channel activity in adult rat and neonatal mouse ventricular myocytes. The alpha1-adrenoceptor agonist methoxamine (MTX) reversibly inhibited the pinacidil-activated K(ATP) current in a concentration-dependent manner (IC50 20.9+/-6.6 micromol/L). This inhibition did not occur when the specific alpha1-adrenoceptor antagonist, prazosin, was present. An involvement of G proteins is suggested by the ability of GDPbetaS to prevent this response. Blockade of PLC by U-73122 (2 micromol/L) or neomycin (2 mmol/L) attenuated the MTX-induced inhibition of K(ATP) channel activity. In contrast, the MTX response was unaffected by protein kinase C inhibition or stimulation by H-7 (100 micro mol/L) or phorbol 12,13-didecanoate. The MTX-induced inhibition became irreversible in the presence of wortmannin (20 micro mol/L), an inhibitor of phosphatidylinositol-4 kinase, which is expected to prevent membrane PIP2 replenishment. In excised inside-out patch membranes, pinacidil induced a significantly rightward shift of ATP sensitivity of the channel. This phenomenon was reversed by pretreatment of myocytes with MTX. Direct visualization of PIP2 subcellular distribution using a PLCdelta pleckstrin homology domain-green fluorescent protein fusion constructs revealed reversible translocation of green fluorescent protein fluorescence from the membrane to the cytosol after alpha1-adrenoceptor stimulation. Our data demonstrate that alpha1-adrenoceptor stimulation reduces the membrane PIP2 level, which in turn inhibits pinacidil-activated K(ATP) channels
— id: 59248, year: 2002, vol: 91, page: 232, stat: Journal Article,

Frequenin as a regulator of Kv4 K+ channels: Mechanisms of action and cardiac expression
Nakamura, TY; Sturn, E; Pountney, DJ; Orenzoff, B; Artman, M; Coetzee, WA
2002 NOV 5 ;106(19):47-48, Circulation
— id: 37199, year: 2002, vol: 106, page: 47, 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,

Kir6.1 K+ channel subunits are expressed in mouse ventricular myocytes and associate with the actin cytoskeleton
Rosner, E; Lopez, G; Porter, LM; Pountney, DJ; Nakamura, TY; Coetzee, WA
2002 JAN ;82(1):589A-589A, Biophysical journal
— id: 55331, year: 2002, vol: 82, page: 589A, stat: Journal Article,

Frequenin as a regulator of Kv4 K+ channels: Cardiac expression and mechanisms of action
Sturm, E; Artman, M; Pountney, D; Coetzee, WA; Nakamura, TY
2002 Apr;51(4):182-, Pediatric research
— id: 27455, year: 2002, vol: 51, page: 182, stat: Journal Article,

Developmental changes in the regulation of cardiac contractile function
Zhou YY; Nakamura TY; Coetzee WA; Artman M
Cardiac development Boston : Kluwer, 2002,
— id: 2800, year: 2002, vol: , page: 133, stat: Chapter,

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,

Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters
Nakamura, T Y; Iwata, Y; Sampaolesi, M; Hanada, H; Saito, N; Artman, M; Coetzee, W A; Shigekawa, M
2001 Aug;281(2):C690-C699, American journal of physiology. Cell physiology
Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex, causes cardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Using cultured myotubes prepared from skeletal muscle of normal and Bio14.6 hamsters (J2N-k strain), we investigated the possibility that the delta-SG deficiency may lead to alterations in ionic conductances, which may ultimately lead to myocyte damage. In cell-attached patches (with Ba(2+) as the charge carrier), an approximately 20-pS channel was observed in both control and Bio14.6 myotubes. This channel is also permeable to K(+) and Na(+) but not to Cl(-). Channel activity was increased by pressure-induced stretch and was reduced by GdCl(3) (>5 microM). The basal open probability of this channel was fourfold higher in Bio14.6 myotubes, with longer open and shorter closed times. This was mimicked by depolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes, the unidirectional basal Ca(2+) influx was enhanced compared with control. This Ca(2+) influx was sensitive to GdCl(3), signifying that stretch-activated cation channels may have been responsible for Ca(2+) influx in Bio14.6 hamster myotubes. These results suggest a possible mechanism by which cell damage might occur in this animal model of muscular dystrophy
— id: 134936, year: 2001, vol: 281, page: C690, 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,

Is the molecular composition of K-ATP channels more complex than originally thought?
Pountney, DJ; Sun, ZQ; Porter, LM; Nitabach, MN; Nakamura, TY; Holmes, D; Rosner, E; Kaneko, M; Manaris, T; Holmes, TC; Coetzee, WA
2001 AUG ;33(8):1541-1546, Journal of molecular & cellular cardiology
ATP-sensitive K+ (K-ATP) channels are abundantly expressed in the heart and may be involved in the pathogenesis of myocardial ischemia, These channels are heteromultimeric, consisting of four pore-forming subunits (Kir6.1, Kir6.2) and four sulfonylurea receptor (SUR) subunits in an octameric assembly. Conventionally, the molecular composition of K-ATP channels in cardiomyocytes and pancreatic beta -cells is thought to include the Kir6.2 subunit and either the SUR2A or SUR1 subunits, respectively. However, Kir6.1 mRNA is abundantly expressed in the heart, suggesting that Kir6.1 and Kir6.2 subunits may co-assemble to form functional heteromeric channel complexes. Here we provide two independent lines of evidence that heteromultimerization between Kir6.1 and Kir6.2 subunits is possible in the presence of SUR2A, We generated dominant negative Kir6 subunits by mutating the GFG residues in the channel pore to a series of alanine residues. The Kir6.1-AAA pore mutant subunit suppressed both wt-Kir6.1/SUR2A and wt-Kir6.2/SUR2A currents in transfected HEK293 cells, Similarly, the dominant negative action of Kir6.2-AAA does not discriminate between either of the wild-type subunits, suggesting, an interaction between Kir6.1 and Kir6.2 subunits within the same channel complex. Biochemical data support this concept: immunoprecipitation with Kir6.1 antibodies also co-precipitates Kir6.2 subunits and conversely immunoprecipitation with Kir6.2 antibodies co-precipitates Kir6.1 subunits. Collectively our data provide direct electrophysiological and biochemical evidence for heteromultimeric assembly between Kir6.1 and Kir6.2, This paradigm has profound implications for understanding the properties of native K-ATP channels in the heart and other tissues. (C) 2001 Academic Press
— id: 54965, year: 2001, vol: 33, page: 1541, stat: Journal Article,

KIR6.1 K+ channel subunits are expressed in mouse ventricular myocytes and associate with the actin cytoskeleton
Rosner, E; Lopez, G; Nakamura, TY; Porter, L; Pountney, DJ; Coetzee, WA
2001 JUN ;33(6):A102-A102, Journal of molecular & cellular cardiology
— id: 54848, year: 2001, vol: 33, page: A102, stat: Journal Article,

Thyroid hormone increases pacemaker activity in rat neonatal atrial myocytes
Sun ZQ; Ojamaa K; Nakamura TY; Artman M; Klein I; Coetzee WA
2001 Apr;33(4):811-824, Journal of molecular & cellular cardiology
The effects of thyroid hormone (3,3',5-triiodo- L -thyronine, T3) on pacemaker activity were studied with electrophysiological and pharmacological approaches using spontaneously beating neonatal atrial myocytes cultured from 2-day-old rats. Treatment with T3 (10(-8)m) for 24-48 h led to a positive chronotropic effect. The beating rate of T3-treated cells was 244+/-19 beats/min and for control cells it was 122+/-10 beats/min (P<0.05). Action potentials were recorded and showed that the predominant effect of T3 was to increase the diastolic depolarization rate (99.5+/-9.8 in T3-treated group v 44.0+/-7.8 mV/s in untreated group). Some cells that exhibited pacemaker activity lacked a pacemaker current (I(f)) under voltage clamp conditions I(f)was recorded in 5 of 12 spontaneously active control cells and in 6 of 10 T3-treated cells. In those cells exhibiting the pacemaker current, the I(f)density was significantly larger in T3-treated cells (-7.9+/-2.6 pA/pF v-1.8+/-0.5 pA/pF in control). The L-type Ca2+ current density was similar in the two groups (at -7 mV, -7.5+/-1.5 in treated group v-8.6+/-1.0 pA/pF in control). In the presence of T3, the Na+-Ca2+ exchanger current (I(Na/Ca)) density was larger (e.g. at +60 mV, it was 4.8+/-0.5 v 3.5+/-0.2 pA/pF in control cells, P<0.05). As intracellular Ca2+ is extruded from the cell, the electrogenic Na+-Ca2+ exchanger causes a declining inward current, which may contribute to the pacemaker potential-this declining inward current was demonstrated using the action potential voltage clamp technique and was shown to be larger in T3-treated myocytes. Our data demonstrate that thyroid hormone enhances pacemaker activity and that this may be due in part to an increased Na+-Ca2+ exchanger activity
— id: 21224, year: 2001, vol: 33, page: 811, 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,

Calcium regulation in the developing heart
Artman M; Haddock PS; Coetzee WA
Etiology and morphogenesis of congential heart disease Armonk NY : Futura Pub., 2000,
— id: 2802, year: 2000, vol: , page: 289, stat: Chapter,

Cellular basis for age-related differences in cardiac excitation-contraction coupling
Artman M; Henry G; Coetzee WA
2000 Sep 1;11(3):185-194, Progress in pediatric cardiology
Clinical experience indicates that infants and young children respond to a variety of cardiovascular pharmacological and physiological interventions differently than adults. What is less clear, however, are the cellular and molecular mechanisms that contribute to these age-related differences. Based largely upon results from animal models, it is apparent that developmental changes occur in numerous pathways and proteins involved in the regulation of contractile function and in the determinants of inotropic responsiveness. The purposes of this review are to provide a brief overview of cardiac excitation-contraction and to illustrate some of the important age-related differences in the mechanisms involved in calcium regulation in the heart. This scientific foundation may help to explain certain clinical observations in the very young. Furthermore, it is hoped that a better understanding of the fundamental processes involved in controlling cardiac contractile function will stimulate additional research in the search for more specific, rational and age-appropriate cardiovascular therapeutics
— id: 39556, year: 2000, vol: 11, page: 185, stat: Journal Article,

Anti-arrhythmic effects of levcromakalim in the ischaemic rat heart: a dual mechanism of action?
Coetzee WA; Wells T; Avkiran M
2000 Aug 25;402(3):263-274, European journal of pharmacology
The action of pharmacological openers of K(ATP) channels depends on the availability and levels of various intracellular nucleotides. Since these are subject to change during myocardial ischaemia, K(ATP) channel openers may affect ischaemic and non-ischaemic tissue differentially. Using a recently developed dual coronary perfusion method, we investigated the effects on arrhythmias of the prototypical K(ATP) channel opener levcromakalim when applied selectively to ischaemic and/or non-ischaemic tissue. A novel perfusion cannula was used to independently perfuse the left and right coronary beds of hearts isolated from rats. Selective infusion of levcromakalim (3, 10 or 30 muM) into the left coronary bed in the absence of ischaemia did not induce ventricular arrhythmias. Regional zero-flow ischaemia was induced by cessation of flow to the left coronary bed and hearts received levcromakalim selectively into either the left, right, or both coronary beds. When applied selectively to the ischaemic left coronary bed, levcromakalim (3, 10 or 30 muM; n=10/group) delayed the onset of ventricular tachycardia in a dose-dependent manner (by 21*, 43* and 112%* at 3, 10 and 30 muM; *P<0.05 vs. control). When applied only to the non-ischaemic right coronary bed, levcromakalim reduced the incidence of ventricular tachycardia during later phases of ischaemia (from 100% in controls to 30%*). When present in both coronary beds, levcromakalim had a striking anti-arrhythmic effect - the overall incidence of ventricular tachycardia being reduced from 100% in controls to 20%*. We conclude that levcromakalim may have an anti-arrhythmic effect when applied either to ischaemic or non-ischaemic tissue but that the mechanisms may differ depending on the metabolic state of the heart
— id: 11530, year: 2000, vol: 402, page: 263, 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,

Overexpression of sorcin in the murine heart modulates cardiac performance
Fischer, A; Meyers, MB; Nakamura, TY; Ai, ZW; Coetzee, WA; Fishman, GI
2000 OCT 31 ;102(18):296-296, Circulation
— id: 55247, year: 2000, vol: 102, page: 296, stat: Journal Article,

Amiodarone inhibits cardiac ATP-sensitive potassium channels
Holmes DS; Sun ZQ; Porter LM; Bernstein NE; Chinitz LA; Artman M; Coetzee WA
2000 Oct;11(10):1152-1158, Journal of cardiovascular electrophysiology
INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone
— id: 39525, year: 2000, vol: 11, page: 1152, stat: Journal Article,

Amiodarone inhibits cardiac ATP-Sensitive K+ channels
Holmes, DS; Sun, ZQ; Porter, L; Artman, M; Chinitz, L; Coetzee, WA
2000 FEB ;35(2):97A-97A, Journal of the American College of Cardiology
— id: 54747, year: 2000, vol: 35, page: 97A, stat: Journal Article,

Modeling sub-cellular calcium gradients during EC coupling in newborn ventricular myocytes
Jafri, MS; Artman, M; Coetzee, WA
2000 JAN ;78(1):455A-455A, Biophysical journal
— id: 54765, year: 2000, vol: 78, page: 455A, 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,

Stretch-activated Ca2+ permeable channels are activated in delta sarcoglycan-deficient myotubes from BIO14.6 hamster
Nakamura, TY; Sampaolesi, M; Iwata, Y; Hanada, H; Coetzee, WA; Artman, M; Shigekawa, M
2000 JAN ;78(1):472A-472A, Biophysical journal
— id: 54766, year: 2000, vol: 78, page: 472A, stat: Journal Article,

Is the Molecular Composition of K(ATP)Channels more Complex than Originally Thought?
Pountney DJ; Sun ZQ; Porter LM; Nitabach MN; Nakamura TY; Holmes D; Rosner E; Kaneko M; Manaris T; Holmes TC; Coetzee WA
2000 Aug;33(8):1541-1546, Journal of molecular & cellular cardiology
ATP-sensitive K(+)(K(ATP)) channels are abundantly expressed in the heart and may be involved in the pathogenesis of myocardial ischemia. These channels are heteromultimeric, consisting of four pore-forming subunits (Kir6.1, Kir6.2) and four sulfonylurea receptor (SUR) subunits in an octameric assembly. Conventionally, the molecular composition of K(ATP)channels in cardiomyocytes and pancreatic beta -cells is thought to include the Kir6.2 subunit and either the SUR2A or SUR1 subunits, respectively. However, Kir6.1 mRNA is abundantly expressed in the heart, suggesting that Kir6.1 and Kir6.2 subunits may co-assemble to form functional heteromeric channel complexes. Here we provide two independent lines of evidence that heteromultimerization between Kir6.1 and Kir6.2 subunits is possible in the presence of SUR2A. We generated dominant negative Kir6 subunits by mutating the GFG residues in the channel pore to a series of alanine residues. The Kir6.1-AAA pore mutant subunit suppressed both wt-Kir6.1/SUR2A and wt-Kir6.2/SUR2A currents in transfected HEK293 cells. Similarly, the dominant negative action of Kir6.2-AAA does not discriminate between either of the wild-type subunits, suggesting an interaction between Kir6.1 and Kir6.2 subunits within the same channel complex. Biochemical data support this concept: immunoprecipitation with Kir6.1 antibodies also co-precipitates Kir6.2 subunits and conversely, immunoprecipitation with Kir6.2 antibodies co-precipitates Kir6.1 subunits. Collectively, our data provide direct electrophysiological and biochemical evidence for heteromultimeric assembly between Kir6.1 and Kir6.2. This paradigm has profound implications for understanding the properties of native K(ATP)channels in the heart and other tissues.
— id: 21130, year: 2000, vol: 33, page: 1541, stat: Journal Article,

Biochemical and electrophysiological evidence for heteromultimeric assembly of Kir6 subfamily members
Pountney, DJ; Sun, ZQ; Porter, L; Nakamura, TY; Nitabach, MN; Kaneko, M; Manaris, T; Rosner, E; Holmes, TC; Artman, M; Coetzee, WA
2000 OCT 31 ;102(18):260-260, Circulation
— id: 55245, year: 2000, vol: 102, page: 260, stat: Journal Article,

Effects of thyroid hormone on action potential and repolarizing currents in rat ventricular myocytes
Sun ZQ; Ojamaa K; Coetzee WA; Artman M; Klein I
2000 Feb;278(2):E302-E307, American journal of physiology. Endocrinology & metabolism
Thyroid hormones play an important role in cardiac electrophysiology through both genomic and nongenomic mechanisms of action. The effects of triiodothyronine (T(3)) on the electrophysiological properties of ventricular myocytes isolated from euthyroid and hypothyroid rats were studied using whole cell patch clamp techniques. Hypothyroid ventricular myocytes showed significantly prolonged action potential duration (APD(90)) compared with euthyroid myocytes, APD(90) of 151 +/- 5 vs. 51 +/- 8 ms, respectively. Treatment of hypothyroid ventricular myocytes with T(3) (0.1 microM) for 5 min significantly shortened APD by 24% to 115 +/- 10 ms. T(3) similarly shortened APD in euthyroid ventricular myocytes, but only in the presence of 4-aminopyridine (4-AP), an inhibitor of the transient outward current (I(to)), which prolonged the APD by threefold. Transient outward current (I(to)) was not affected by the acute application of T(3) to either euthyroid or hypothyroid myocytes; however, I(to) density was significantly reduced in hypothyroid compared with euthyroid ventricular myocytes
— id: 8549, year: 2000, vol: 278, page: E302, stat: Journal Article,

Mechanism(s) underlying increased pacemaker activity of rat neonatal atrial myocytes thyroid hormone
Sun, ZQ; Ojamaa, K; Artman, M; Klein, I; Coetzee, WA
2000 JAN ;78(1):224A-224A, Biophysical journal
— id: 54763, year: 2000, vol: 78, page: 224A, 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,

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,

Subcellular [Ca2+]i gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes
Haddock PS; Coetzee WA; Cho E; Porter L; Katoh H; Bers DM; Jafri MS; Artman M
1999 Sep 3;85(5):415-427, Circulation research
The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca2+]i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca2+]i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca2+]i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 micromol/L thapsigargin) slowed the rate of rise and decline of Ca2+ in AD myocytes but did not alter Ca2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca2+ release events ('Ca2+ sparks') occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na(+)-Ca2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na(+)-Ca2+ exchanger during an action potential can account for the subsarcolemmal Ca2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release during normal EC coupling in NB ventricular myocytes
— id: 6195, year: 1999, vol: 85, page: 415, stat: Journal Article,

Spatial organization of excitation-contraction coupling microarchitecture in developing rabbit ventricular myocytes
Haddock, PS; Coetzee, WA; Artman, M
1999 JAN ;76(1):A460-A460, Biophysical journal
— id: 54002, year: 1999, vol: 76, page: A460, stat: Journal Article,

Mechanisms of EC coupling in newborn rabbit heart
Haddock, PS; Patel, A; Artman, M; Coetzee, WA
1999 JAN ;76(1):A460-A460, Biophysical journal
— id: 54001, year: 1999, vol: 76, page: A460, 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,

Sources of activator calcium in the developing rabbit heart
Patel, A; Haddock, PS; Coetzee, WA; Artman, M
1999 APR ;45(4):58A-58A, Pediatric research
— id: 54065, year: 1999, vol: 45, page: 58A, 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,

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,

Non-genomic effects of thyroid hormone on a plateau K current in rat ventricular myocytes
Sun, KQ; Ojamaa, K; Artman, M; Klein, I; Coetzee, WA
1999 JAN ;76(1):A367-A367, Biophysical journal
— id: 53999, year: 1999, vol: 76, page: A367, stat: Journal Article,

Spatial and temporal changes in [Ca](i) in immature rabbit ventricular myocytes
Cho, E; Haddock, PS; Coetzee, WA; Bers, DM; Artman, M
1998 FEB ;74(2):A271-A271, Biophysical journal
— id: 53439, year: 1998, vol: 74, page: A271, stat: Journal Article,

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes
Haddock PS; Artman M; Coetzee WA
1998 May;435(6):789-795, Pflugers archiv = European journal of physiology
Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth
— id: 7590, year: 1998, vol: 435, page: 789, stat: Journal Article,

Is the Na-Ca exchanger involved in apparent depolarization-induced calcium release in cardiac myocytes?
Haddock, PS; Coetzee, WA; Artman, M
1998 FEB ;74(2):A267-A267, Biophysical journal
— id: 53438, year: 1998, vol: 74, page: A267, 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,

Role of the sarcoplasmic reticulum in contraction and relaxation of immature rabbit ventricular myocytes
Balaguru D; Haddock PS; Puglisi JL; Bers DM; Coetzee WA; Artman M
1997 Oct;29(10):2747-2757, Journal of molecular & cellular cardiology
Previous indirect studies of newborn hearts have suggested a diminished functional role of the SR and a greater dependency upon trans-sarcolemmal Ca2+ fluxes to directly elicit contraction and promote relaxation. We tested the hypothesis that the SR in newborn rabbit hearts is functionally incompetent by measuring contraction and relaxation in ventricular myocytes isolated from the hearts of 1-2-day-old (newborn), 10-12-day-old (juvenile) and >150-day-old (adult) rabbits. Electrically stimulated twitch characteristics were compared to those elicited by the rapid application of 10 mm caffeine in the presence and absence of functional sarcolemmal Na-Ca exchange (disabled using a Na+- and Ca2+-free extracellular solution). During steady state, electrically-induced contractions were lower in amplitude in newborn and juvenile compared to adult myocytes (2.9+/-0.5 and 3.4+/-0.3 v 8.5+/-0.9% of resting cell length, respectively; n=24-29) and relaxation was slower in immature myocytes (t0.75 values: newborn 250+/-20; juvenile 240+/-10; adult 130+/-20 ms, n=14-21). Contrary to our hypothesis, caffeine triggered sufficient SR Ca2+ release from immature myocytes to elicit contractions of similar magnitude to adults (newborn 12.8+/-1. 1; juvenile 14.0+/-0.9; adult 15.0+/-1.6% of resting cell length, n=25-29). The amplitude of indo-1 Ca2+ transients during steady-state twitch was 36+/-12% of the maximal caffeine-induced Ca2+ transient in newborns (n=6) and 59+/-4% in adults (n=6). Caffeine slightly prolonged relaxation in adult myocytes (t0. 75=200+/-30 ms), but accelerated relaxation in newborn and juvenile myocytes (t0.75=180+/-20 and 150+/-30 ms, respectively). When both the SR and Na-Ca exchanger were disabled, the rate of relaxation (attributable to the sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ uniporter) of newborn and juvenile myocytes was significantly faster than in the adults (1660+/-210 and 3030+/-180 v 4530+/-310 ms, respectively; n=14-21). We conclude that neonatal and adult rabbit ventricular myocytes have comparable SR Ca2+ load, but neonatal cells exhibit smaller fractional SR Ca2+ release during steady-state contractions and greater Ca2+ removal by sarcolemmal Na-Ca exchange during relaxation.
— id: 8376, year: 1997, vol: 29, page: 2747, stat: Journal Article,

Na+/Ca2+ exchange current and contractions measured under Cl(-)-free conditions in developing rabbit hearts
Haddock PS; Coetzee WA; Artman M
1997 Aug;273(2 Pt 2):H837-H846, American journal of physiology. Heart & circulatory physiology
Previous studies suggesting a greater functional role of cardiac Na+/Ca2+ exchange at birth were performed using tightly buffered free cytosolic Ca2+ concentration ([Ca2+]i). Because Na+/Ca2+ exchange current (INaCa) is influenced by physiological fluctuations in [Ca2+]i, we used conditions of minimally buffered [Ca2+]i to simultaneously record INaCa and cell contractions in single ventricular myocytes isolated from 1 to 27-day-old and adult rabbits. With conventional Cl(-)-containing solutions. Ni(2+)-sensitive outward and inward charge movements were unbalanced, suggesting the presence of a contaminating current (presumably the Ca(2+)-activated Cl- current). Removing Cl- abolished this discrepancy in all age groups and allowed for the accurate quantitation of INaCa. Under Cl(-)-free conditions, outward and inward charge movements were high at birth (4 days: 0.42 +/- 0.03 and -0.38 +/- 0.04 pC/pF, respectively) and decreased postnatally (adult: 0.08 +/- 0.01 and -0.07 +/- 0.01 pC/pF, respectively). Newborn but not adult myocytes contracted during depolarizations in the presence of nifedipine, ryanodine, and thapsigargin. The magnitudes of outward charge movement (Ca2+ influx) and cell shortening exhibited similar voltage dependence, consistent with INaCa-mediated contractions. These results indicate that INaCa can directly support contraction in newborn rabbit ventricular myocytes
— id: 7162, year: 1997, vol: 273, page: H837, stat: Journal Article,

Action potential voltage clamp of Na-Ca exchange current: Age-dependent changes in rabbit ventricle
Haddock, PS; Artman, M; Coetzee, WA
1997 FEB ;72(2):MP238-MP238, Biophysical journal
— id: 53306, year: 1997, vol: 72, page: MP238, stat: Journal Article,

Na-Ca exchange inhibitory peptide prevents apparent depolarization induced calcium release in rat ventricular myocytes
Haddock, PS; Artman, M; Coetzee, WA
1997 OCT 21 ;96(8):991-991, Circulation
— id: 105047, year: 1997, vol: 96, page: 991, 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,

Ammonium chloride decreases a voltage-sensitive potassium current in isolated coronary smooth muscle myocytes
Webster, RG; Coetzee, WA; Clapp, LH
1997 FEB ;72(2):THAM9-THAM9, Biophysical journal
— id: 53310, year: 1997, vol: 72, page: THAM9, 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,

Cardiac sarcoplasmic reticulum in neonates can release calcium comparable to adult myocytes
Balaguru, D; Haddock, PS; Coetzee, WA; Artman, M
1996 OCT 15 ;94(8):2824-2824, Circulation
— id: 52745, year: 1996, vol: 94, page: 2824, stat: Journal Article,

Ontogeny and hormonal regulation of cardiac Na(+)-Ca2+ exchanger expression in rabbits
Boerth SR; Coetzee WA; Artman M
1996 Apr 15;779:536-538, Annals of the New York Academy of Sciences
— id: 6984, year: 1996, vol: 779, page: 536, stat: Journal Article,

Ontogeny of forward and reverse mode Na-Ca exchange in the developing rabbit heart
Haddock, PS; Coetzee, WA; Artman, M
1996 OCT 15 ;94(8):687-687, Circulation
— id: 52742, year: 1996, vol: 94, page: 687, stat: Journal Article,

Na+/Ca2+ exchange current density in cardiac myocytes from rabbits and guinea pigs during postnatal development
Artman M; Ichikawa H; Avkiran M; Coetzee WA
1995 Apr;268(4 Pt 2):H1714-H1722, American journal of physiology. Heart & circulatory physiology
It has been proposed that the activity of the cardiac sarcolemmal Na+/Ca2+ exchanger may be greatest in developing animals before the sarcoplasmic reticulum (SR) reaches functional maturity. Experiments were performed in rabbits, which have a sparse SR at birth, and in newborn guinea pigs, which exhibit a more extensive SR. Whole cell voltage clamp techniques were used to characterize the Ni(2+)-sensitive Na+/Ca2+ exchange current in single freshly isolated cardiac myocytes. Na+/Ca2+ exchange current was measured from a holding potential of -40 mV by using a slow-ramp voltage protocol (-120 to +60 mV, 0.09 V/s) in the presence of Ba2+, Cs+, tetraethylammonia, D-600, and ouabain to block Ca2+, Na+, and K+ currents. Experiments in developing rabbits (1-22 days old) demonstrated that Na+/Ca2+ exchange current density was greatest at 1-4 days and declined rapidly over the first 3 wk of age. In contrast, Na+/Ca2+ exchange current density in newborn guinea pig myocytes did not differ from that recorded in adults. These results confirm that Na+/Ca2+ exchange is functional at birth in both rabbits and guinea pigs. The species-related difference in the ontogeny of Na+/Ca2+ exchange is consistent with the concept that Na+/Ca2+ exchange assumes a relatively greater role in newborn animals with a sparse SR
— id: 8294, year: 1995, vol: 268, page: H1714, stat: Journal Article,

Effects of thiol-modifying agents on KATP channels in guinea pig ventricular cells
Coetzee, W A; Nakamura, T Y; Faivre, J F
1995 Nov;269(5 Pt 2):H1625-H1633, American journal of physiology
ATP-sensitive K+ (KATP) channels are thought only to open during conditions of metabolic impairment (e.g., myocardial ischemia). However, the regulation of KATP channel opening during ischemia remains poorly understood. We tested whether thiol (SH) group oxidation, which is known to occur during ischemia, may be involved in KATP channel regulation. Inside-out membrane patches were voltage clamped at a constant potential (O mV) in asymmetrical K+ solutions. The effects of compounds that specifically modify SH groups [p-chloromercuri-phenylsulfonic acid (pCMPS), 5-5'-dithio-bis(2-nitrobenzoic acid) [DTNB], and thimerosal] were tested. The membrane-impermeable compound, pCMPS (> or = 5 microM), caused a quick and irreversible inhibition of KATP channel activity. The reducing agent, dl-dithiothreitol (DTT) (3 mM) was able to reverse this inhibition. DTNB (500 microM) caused a rapid, but spontaneously reversible, block of KATP channel activity. After DTNB, no change was observed in single channel conductance. Oxidized glutathione (GSSG, 3 mM) did not block KATP channel activity. Thimerosal (100-500 microM) induced a DTT-reversible block of partially rundown KATP channels, or channels that underwent complete rundown; these channels were reactivated with trypsin (1 mg/ml). Thimerosal did not block KATP channels that had a high degree of activity. However, the ATP sensitivity was decreased; the concentration of ATP needed to half-maximally inhibit the channel (Ki) was increased from 47 +/- 12 to 221 +/- 35 microM (n = 6, P < 0.05). This was not due to a spontaneous change with time.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 135031, year: 1995, vol: 269, page: H1625, stat: Journal Article,

ATP DEPLETION WITH OR WITHOUT ALPHA-RECEPTOR OCCUPATION IS INSUFFICIENT TO PRECONDITION ISOLATED RAT CARDIAC MYOCYTES
KAYA, B; COETZEE, WA; SHATTOCK, MJ
1995 OCT 15 ;92(8):3449-3449, Circulation
— id: 105049, year: 1995, vol: 92, page: 3449, stat: Journal Article,

SPONTANEOUS AND THIOL GROUP-INDUCED CHANGES IN ATP-SENSITIVITY OF K-ATP CHANNELS FROM ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES
NAKAMURA, TY; FAIVRE, JF; COETZEE, WA
1995 FEB ;483P(4):P10-P10, Journal of physiology
— id: 105050, year: 1995, vol: 483P, page: P10, stat: Journal Article,

NA+-CA2+ EXCHANGE CURRENT-DENSITY DECLINES DURING EARLY POSTNATAL MATURATION IN RABBIT VENTRICULAR MYOCYTES
ARTMAN, M; COETZEE, WA
1994 APR ;35(4):A29-A29, Pediatric research
— id: 105052, year: 1994, vol: 35, page: A29, stat: Journal Article,

Oxidant stress inhibits Na-Ca-exchange current in cardiac myocytes: mediation by sulfhydryl groups?
Coetzee, W A; Ichikawa, H; Hearse, D J
1994 Mar;266(3 Pt 2):H909-H919, American journal of physiology
The effects of oxidant stress (xanthine oxidase plus hypoxanthine or photoactivation of rose bengal) on the Na(+)-Ca(2+)-exchange current were studied in guinea pig ventricular myocytes with the use of voltage-clamp techniques. Oxidant stress depressed both the Ni(2+)-sensitive and extracellular calcium concentration ([Ca2+]o)-activated current in a time-dependent manner (e.g., xanthine oxidase plus hypoxanthine inhibited the Ni(2+)-sensitive current at +60 mV from 6.81 +/- 3.24 to 5.54 +/- 0.48 pA/pF; n = 6; P < 0.05). This effect was independent of the [Ca2+] of the pipette solution. Diamide, an alkylating agent that modifies protein sulfhydryl groups, also decreased the Ni(2+)-sensitive current (at + 60 mV: from 5.76 +/- 1.55 to 3.43 +/- 0.99 pA/pF; n = 6; P < 0.05). The stoichiometry (n) and partition coefficient (gamma) of the electrogenic Na(+)-Ca(2+)-exchange current seemed unchanged. Our results suggest that oxidant stress causes a direct or indirect sulfhydryl group-mediated decrease of the Na(+)-Ca2+ exchanger
— id: 134945, year: 1994, vol: 266, page: H909, stat: Journal Article,

FREE-RADICALS AND REPERFUSION ARRHYTHMIAS
COETZEE, WA
1994 MAR ;28(3):425-426, Cardiovascular research
— id: 105053, year: 1994, vol: 28, page: 425, stat: Journal Article,

Effects of R-56865 on transient inward current, Na(+)-Ca2+ exchange, and Ca2+ release from SR in cardiac myocytes
Ichikawa, H; Hearse, D J; Coetzee, W A
1994 Feb;266(2 Pt 2):H511-H520, American journal of physiology
Voltage-clamp studies were performed on guinea pig ventricular myocytes to clarify the action of N-(1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl)-N-methyl-2-benzothiazo lamine (R-56865), an inhibitor of cardiac glycoside-induced arrhythmias. Transient inward current ((Iti)) was induced using low-K+/high-Ca2+ Tyrode solution. R-56865 (1 mM) was found to abolish I(ti). R-56865 had no influence on the peak Ca2+ current, steady-state current during the clamp, holding current, or the Ni(2+)-sensitive electrogenic Na(+)-Ca2+ exchange current. Fluorescence transients after repolarization (temporally related to the I(ti)) were abolished by R-56865 without affecting the fluorescence transients during depolarization. In separate experiments, the threshold of Ca2+ release from sarcoplasmic reticulum (SR) by the Ca2+ current was found to be unchanged, whereas Ca2+ transients (presumably triggered by Ca2+ entry through the Na(+)-Ca2+ exchanger) were depressed. Our results suggest that R-56865 inhibits spontaneous Ca2+ release from the SR when it is mediated by Ca2+ entry through the Na(+)-Ca2+ exchanger but that it has no direct effect on the well-known 'physiological' Ca(2+)-induced Ca(2+)-release mechanism from SR
— id: 134937, year: 1994, vol: 266, page: H511, stat: Journal Article,

"EFFECTS OF R-56865 ON TRANSIENT INWARD CURRENT, NA+-CA2+ EXCHANGE, AND CA2+ RELEASE FROM SR IN CARDIAC MYOCYTES (VOL 266, PG H511, 1994)"
ICHIKAWA, H; HEARSE, DJ; COETZEE, WA
1994 AUG ;267(2):CP4-CP4, American journal of physiology
— id: 105051, year: 1994, vol: 267, page: CP4, stat: Journal Article,

DEVELOPMENTAL-CHANGES IN THE ELECTROGENIC NA+-CA2+ EXCHANGE CURRENT-DENSITY IN VOLTAGE-CLAMPED, ISOLATED RABBIT VENTRICULAR MYOCYTES
COETZEE, WA; AVKIRAN, M; HEARSE, DJ; ARTMAN, M
1993 MAR ;473(3):P181-P181, Journal of physiology
— id: 105054, year: 1993, vol: 473, page: P181, stat: Journal Article,

ATP-SENSITIVE POTASSIUM CHANNELS AND MYOCARDIAL-ISCHEMIA - WHY DO THEY OPEN
COETZEE, WA
1992 JUN ;6(3):201-208, Cardiovascular drugs & therapy
There is evidence that the 'ATP-sensitive' potassium channel opens, at least during the early stages of myocardial ischemia, despite relatively high ATP levels. Thus, channel opening may partially contribute to potassium efflux and accumulation of extracellular potassium, but probably much more profoundly to electrical abnormalities associated with ischemia, including the development of lethal arrhythmias. Several factors are discussed that may promote a significant open-channel probability of the channel, in spite of relatively high levels of ATP. It is argued that, even with a very low open probability, the magnitude of total membrane current carried by these channels may be substantial (comparable to other potassium currents) because of the high density and conductance of the ATP-sensitive potassium channel. Finally, it is shown how the ATP-sensitive potassium channel may play a role in various tissue types, ranging from the physiological to the pathophysiological. This potassium channel is therefore increasingly targeted for drug development and research
— id: 105059, year: 1992, vol: 6, page: 201, stat: Journal Article,

REGULATION OF ATP SENSITIVE POTASSIUM CHANNEL OF ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES BY SARCOLEMMAL MONOCARBOXYLATE TRANSPORT
COETZEE, WA
1992 NOV ;26(11):1077-1086, Cardiovascular research
Objective: The aim was to describe the effects of extracellular application of monocarboxylates (pyruvate, lactate, or acetate) on current through K(ATP) channels (i(K,ATP)) in isolated guinea pig ventricular myocytes. Methods: The i(K-ATP) was elicited during whole cell voltage clamping by application of metabolic poisons, 2,4 dinitrophenol (150 muM) or glucose free cyanide (1 mM) and could be blocked by glibenclamide (3 muM) Results: Extracellular application of monocarboxylates, pyruvate (0.1-10 MM), L-lactate (0.1-10 mM), and acetate (10 mM) led to a rapid inhibition of i(K,ATP) - an effect which was fully reversible upon washout. Substances without any effect on i(K,ATP) were (10 mM each) gluconate, citrate, glutamate, creatine, succinate, and glycine. The mechanism underlying the effects of monocarboxylates on i(K,ATP) was unlikely to be related to an increased ATP production, since D-lactate (10 mM) essentially had the same effect on i(K,ATP) as the L-isomer of lactate. Furthermore, with intracellular dialysis of alpha-cyano-4-hydroxycinnamate (0.1-0.5 mM), which inhibits pyruvate uptake into mitochondria, extracellular pyruvate exerted the same inhibitory effect on i(K,ATP). High concentrations of extracellular alpha-cyano-4-hydroxycinnamate (4 mM), which blocks the sarcolemmal monocarboxylate carrier, prevented the effects on i(K,ATP) by pyruvate, L-lactate, D-lactate, and acetate. Furthermore, intracellular dialysis with D-lactate (10 mM) led to a more rapid onset of i(K,ATP) when activated by ATP free dialysis. Activity of isolated K(ATP) channels, measured in isolated membrane patches in the inside out or outside out configuration, typically had a single channel conductance of around 80 pS and was blocked by glibenclamide (3-9 muM). No significant effect of pyruvate was observed in either patch configuration. Conclusions: In cardiac tissue there may be some modulatory role involving monocarboxylate transport on K(ATP) channel activity, the nature of which is unclear at present but which may involve cytosolic pH changes. Physiological and pathophysiological implications of these findings are discussed
— id: 105055, year: 1992, vol: 26, page: 1077, stat: Journal Article,

EFFECTS OF OXYGEN FREE-RADICALS ON ISOLATED CARDIAC MYOCYTES FROM GUINEA-PIG VENTRICLE - ELECTROPHYSIOLOGICAL STUDIES
COETZEE, WA; OPIE, LH
1992 JUN ;24(6):651-663, Journal of molecular & cellular cardiology
— id: 105058, year: 1992, vol: 24, page: 651, stat: Journal Article,

EFFECTS OF THE ACE INHIBITOR, PERINDOPRILAT, AND OF ANGIOTENSIN-II ON THE TRANSIENT INWARD CURRENT OF GUINEA-PIG VENTRICULAR MYOCYTES
ENOUS, R; COETZEE, WA; OPIE, LH
1992 JAN ;19(1):17-23, Journal of cardiovascular pharmacology
Hypothetically, certain ischemic and reperfusion arrhythmias may result from the activity of the calcium-dependent transient inward current. The effects of the angiotensin converting enzyme inhibitor, perindoprilat, on the transient inward current of guinea pig ventricular myocytes were studied. The transient inward current was evoked by superfusing the cell with a modified Tyrode's solution (5-4 MM CaCl2 and 0.54 mM KCl). Repetitive voltage clamp steps from a holding potential of -55 to +20 mV (1,000 ms, 0.1 Hz) were applied while dialyzing the cell internally. When administered simultaneously with the change over to the low K+ high Ca2+ solution, perindoprilat (1-mu-M decreased the transient inward current from -9.55 +/- 0.31 to -3.24 +/- 0.24-mu-A/cm2 (p < 0.05). A similar decrease was observed when perindoprilat was administered after first inducing the transient inward current. Perindoprilat also protected from the effects of norepinephrine (0.01 and 0.1-mu-M, which increased the amplitude of the transient inward current from -9.76 +/- 0.17 and -9.99 +/- 0.32-mu-A/cm2 at the end of the 15-min control period to -11.13 +/- 0.67 and -12.67 +/- 0.49-mu-A/cm2, respectively (p < 0.05). The effects of perindoprilat were independent of angiotensin II, which in this preparation decreased the transient inward current. Based on our results, we conclude that perindoprilat decreases the transient inward current and prevents the action of norepinephrine on the transient inward current. The direct effect of the angiotensin converting enzyme inhibitor observed on the transient inward current might explain why angiotensin converting enzyme inhibitors reduce calcium-dependent ouabain-induced or reperfusion arrhythmias
— id: 105060, year: 1992, vol: 19, page: 17, stat: Journal Article,

DOES MODULATION OF INTRACELLULAR PH DURING EARLY REPERFUSION INFLUENCE VULNERABILITY TO REPERFUSION ARRHYTHMIAS
IBUKI, C; COETZEE, WA; HEARSE, DJ; AVKIRAN, M
1992 AUG ;24(11):S32-S32, Journal of molecular & cellular cardiology
— id: 105057, year: 1992, vol: 24, page: S32, stat: Journal Article,

MODULATION OF ELECTROGENIC NA/CA EXCHANGE CURRENT BY OXYGEN RADICAL GENERATING SYSTEMS IN ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES
ICHIKAWA, H; COETZEE, WA; SHATTOCK, MJ; HEARSE, DJ
1992 AUG ;24(11):S28-S28, Journal of molecular & cellular cardiology
— id: 105056, year: 1992, vol: 24, page: S28, stat: Journal Article,

Empleo clinico de los farmacos antagonistas de los canales del calcio = [Clinical use of calcium channel antogonist drugs]
Opie, Lionel H; Coetzee, William A.
Madrid : CEA, 1992,
Traducido al espanol por el Dr. Eduardo de Teresa
— id: 1977, year: 1992, vol: , page: , stat: ,

TRIMETAZIDINE - EFFECTS ON DELAYED AFTERDEPOLARIZATIONS (DADS) AND UPSTROKE VELOCITY OF THE ACTION-POTENTIAL
COETZEE, WA; ENOUS, R; OPIE, LH
1990 AUG ;4(1):806-807, Cardiovascular drugs & therapy
— id: 105061, year: 1990, vol: 4, page: 806, stat: Journal Article,

REPERFUSION DAMAGE - FREE-RADICALS MEDIATE DELAYED MEMBRANE-CHANGES RATHER THAN EARLY VENTRICULAR ARRHYTHMIAS
COETZEE, WA; OWEN, P; DENNIS, SC; SAMAN, S; OPIE, LH
1990 FEB ;24(2):156-164, Cardiovascular research
— id: 105063, year: 1990, vol: 24, page: 156, stat: Journal Article,

EFFECTS OF PROTON BUFFERING AND OF AMILORIDE DERIVATIVES ON REPERFUSION ARRHYTHMIAS IN ISOLATED RAT HEARTS - POSSIBLE EVIDENCE FOR AN ARRHYTHMOGENIC ROLE OF NA+-H+ EXCHANGE
DENNIS, SC; COETZEE, WA; CRAGOE, EJ; OPIE, LH
1990 APR ;66(4):1156-1159, Circulation research
— id: 105062, year: 1990, vol: 66, page: 1156, stat: Journal Article,

REDUCTION OF ISCHEMIC K+ LOSS AND ARRHYTHMIAS IN RAT HEARTS - EFFECT OF GLIBENCLAMIDE, A SULFONYLUREA
KANTOR, PF; COETZEE, WA; CARMELIET, EE; DENNIS, SC; OPIE, LH
1990 FEB ;66(2):478-485, Circulation research
— id: 105064, year: 1990, vol: 66, page: 478, stat: Journal Article,

EFFECTS OF CORONARY FLOW, PACING RATE, ISOPRENALINE AND DILTIAZEM ON ISCHEMIC VENTRICULAR ARRHYTHMIAS IN GUINEA-PIG HEARTS
DENNIS, SC; COETZEE, WA; DEJONG, JW; CLUSIN, W; OPIE, LH
1989 JAN ;248(1):372-377, Journal of pharmacology & experimental therapeutics
— id: 105066, year: 1989, vol: 248, page: 372, stat: Journal Article,

Clinical use of calcium channel antagonist drugs
Opie, Lionel H; Coetzee, William A
Boston MA : Kluwer, 1989,
— id: 1563, year: 1989, vol: , page: , stat: ,

THE EFFECT OF ENERGY-METABOLISM ON THE TRANSIENT INWARD CURRENT OF VENTRICULAR MYOCYTES
COETZEE, W; BIERMANS, G; CALLEWAERT, G; VEREECKE, J; OPIE, L; CARMELIET, E
1988 JUN ;84(6):529-529, South African journal of science
— id: 105068, year: 1988, vol: 84, page: 529, stat: Journal Article,

THE EFFECT OF INHIBITION OF MITOCHONDRIAL ENERGY-METABOLISM ON THE TRANSIENT INWARD CURRENT OF ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES
COETZEE, W; BIERMANS, G; CALLEWAERT, G; VEREECKE, J; OPIE, LH; CARMELIET, E
1988 MAR ;20(3):181-185, Journal of molecular & cellular cardiology
— id: 105069, year: 1988, vol: 20, page: 181, stat: Journal Article,

ROLE OF CALCIUM-IONS IN REPERFUSION ARRHYTHMIAS - RELEVANCE TO PHARMACOLOGIC INTERVENTION
OPIE, LH; COETZEE, WA
1988 DEC ;2(5):623-636, Cardiovascular drugs & therapy
— id: 105065, year: 1988, vol: 2, page: 623, stat: Journal Article,

A POTENTIAL ROLE OF CALCIUM-IONS IN EARLY ISCHEMIC AND REPERFUSION ARRHYTHMIAS
OPIE, LH; COETZEE, WA; DENNIS, SC; THANDROYEN, FT
1988 MAR 31 ;522(1):464-477, Annals of the New York Academy of Sciences
— id: 105067, year: 1988, vol: 522, page: 464, stat: Journal Article,

INHIBITION BY SIMULATED ISCHEMIA OR HYPOXIA OF DELAYED AFTERDEPOLARIZATIONS PROVOKED BY CYCLIC-AMP - SIGNIFICANCE FOR ISCHEMIC AND REPERFUSION ARRHYTHMIAS
SAMAN, S; COETZEE, WA; OPIE, LH
1988 FEB ;20(2):91-95, Journal of molecular & cellular cardiology
— id: 105070, year: 1988, vol: 20, page: 91, stat: Journal Article,

CALCIUM-CHANNEL BLOCKERS AND EARLY ISCHEMIC VENTRICULAR ARRHYTHMIAS - ELECTROPHYSIOLOGICAL VERSUS ANTIISCHEMIC EFFECTS
COETZEE, WA; DENNIS, SC; OPIE, LH; MULLER, CA
1987 MAY ;19(2):77-97, Journal of molecular & cellular cardiology
— id: 105076, year: 1987, vol: 19, page: 77, stat: Journal Article,

EFFECTS OF COMPONENTS OF ISCHEMIA AND METABOLIC INHIBITION ON DELAYED AFTERDEPOLARIZATIONS IN GUINEA-PIG PAPILLARY-MUSCLE
COETZEE, WA; OPIE, LH
1987 AUG ;61(2):157-165, Circulation research
— id: 105075, year: 1987, vol: 61, page: 157, stat: Journal Article,

PROPOSED ROLE OF ENERGY SUPPLY IN THE GENESIS OF DELAYED AFTERDEPOLARIZATIONS - IMPLICATIONS FOR ISCHEMIC OR REPERFUSION ARRHYTHMIAS
COETZEE, WA; OPIE, LH; SAMAN, S
1987 OCT ;19(2):13-21, Journal of molecular & cellular cardiology
— id: 105071, year: 1987, vol: 19, page: 13, stat: Journal Article,

MODULATION OF THE TRANSIENT INWARD CURRENT BY METABOLIC INHIBITION AND INTRACELLULAR ATP - IMPLICATIONS FOR ISCHEMIC ARRHYTHMIAS
COETZEE, WA; SCAMPS, F; CARMELIET, EE; OPIE, LH
1987 OCT ;76(4):16-16, Circulation
— id: 105072, year: 1987, vol: 76, page: 16, stat: Journal Article,

REPERFUSION ARRHYTHMIAS - EVIDENCE FOR A ROLE FOR NA+/H+ EXCHANGE AND AN ANTIARRHYTHMIC EFFECT OF AMILORIDE
DENNIS, SC; COETZEE, WA; SIKO, L; OPIE, LH
1987 OCT ;76(4):56-56, Circulation
— id: 105074, year: 1987, vol: 76, page: 56, stat: Journal Article,

EFFECTS OF GLIBENCLAMIDE ON ISCHEMIC ARRHYTHMIAS
KANTOR, PF; COETZEE, WA; DENNIS, SC; OPIE, LH
1987 OCT ;76(4):17-17, Circulation
— id: 105073, year: 1987, vol: 76, page: 17, stat: Journal Article,

MULTIPLE SITES OF MODULATION OF CALCIUM-ION MOVEMENTS IN CARDIAC TISSUE - IMPLICATIONS FOR CARDIAC-ARRHYTHMIAS
COETZEE, WA; OPIE, LH; THANDROYEN, FT
1986 JUN ;18(8):19-19, Journal of molecular & cellular cardiology
— id: 105078, year: 1986, vol: 18, page: 19, stat: Journal Article,

EFFECTS OF COMPONENTS OF SIMULATED ISCHEMIA ON DELAYED AFTER-DEPOLARIZATIONS OF GUINEA-PIG VENTRICULAR MUSCLE
COETZEE, WA; YON, ET; OPIE, LH
1986 AUG ;82(8):443-443, South African journal of science
— id: 105077, year: 1986, vol: 82, page: 443, stat: Journal Article,

ARRHYTHMIAS INDUCED BY REPERFUSION AFTER SIMULATED ISCHEMIA IN GUINEA-PIG PAPILLARY-MUSCLE
COETZEE, WA; SAMAN, S; YON, ET; OPIE, LH
1985 JUN ;81(7):403-403, South African journal of science
— id: 105080, year: 1985, vol: 81, page: 403, stat: Journal Article,

FAILURE OF FREE OXYGEN RADICALS TO ALTER CARDIAC ACTION-POTENTIAL AND REOXYGENATION AUTOMATICITY
DENNIS, SC; COETZEE, WA; YON, E; OPIE, LH
1985 JUN ;72(4):228-228, Circulation
— id: 105079, year: 1985, vol: 72, page: 228, stat: Journal Article,

PACEMAKER ACTIVITY IN RABBIT CARDIAC PURKINJE-FIBERS
CARMELIET, E; COETZEE, W; MUBAGWA, K
1983 JUN ;334(JAN):P55-P56, Journal of physiology
— id: 105081, year: 1983, vol: 334, page: P55, stat: Journal Article,

CATECHOLAMINES AND ELECTROGENIC NA PUMP IN RABBIT CARDIAC PURKINJE-FIBERS
COETZEE, WA; CARMELIET, E
1982 JUN ;80(6):A14-A14, Journal of general physiology
— id: 105082, year: 1982, vol: 80, page: A14, stat: Journal Article,