BACKGROUND

Introduction

The EAG family
Introduction

M-currents are believed to play critical roles in the subthreshold behavior, and response to synaptic inputs of many CNS neurons. M-currents were first described in peripheral sympathetic neurons and have since been identified in neurons of the hippocampus, neocortex and olfactory bulb. Characteristic features of the classical M-current include a relatively low activation voltage, no measurable inactivation, and a low sensitivity to commonly used K+ channel blockers (4-AP and TEA). Another defining feature of the M-current is its inhibition by Ach via muscarinic receptors. It has been suggested that Kcnq2 and Kcnq3 subunits, when found in heteromultimers, are responsible for the classical M-current in sympathetic ganglia. However it is still unclear whether these genes are the molecular components for all M-like currents.

The members of the three subfamilies (eag, erg and elk) of the ether-a-go-go (EAG) family of potassium channel pore forming subunits express currents which, like the M-current, could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. These properties include a low voltage of activation and the ability to carry steady state currents in sub-threshold ranges (see Figure below).

A non-radioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the 8 known EAG family subunits in rat brain was performed in order to identify neuronal populations where the physiological roles of EAG channels could be studied. These distributions were compared to those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus, however only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2 and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses.
The EAG K+ Channel Gene Family
The EAG (ether-a-go-go) family of potassium channels was named after the EAG locus in Drosophila, which when mutated, results in an ether-sensitive leg-shaking phenotype (Ganetzky et al., 1999; Kaplan and Trout, 1969; Warmke et al., 1991). Using methods similar to those utilized to expand the Kv family (e.g. low stringency screens, degenerate RT-PCR, and database screening), 8 mammalian EAG-family genes have been identified (Figure, left). These can be classified in 3 subfamilies which have been named: eag, erg (EAG related gene); and elk (EAG- like K channel). Members within the same subfamily share about 50-70% overall amino acid identity, whereas members of different subfamilies share only 30-40% (see Figure1).
Kv, KCNQ and EAG proteins are members of the S4 superfamily of proteins which also includes the ISK and Slo families, components of Ca2+-activated K+ channels, as well as the components of cyclic-nucleotide-gated channels, the a subunits of voltage-gated Na+ and Ca2+ channels, and the components of pacemaker (Ih) channels. K+ channel proteins of the S4 superfamily consist of a core membrane region consisting of six putative membrane-spanning domains designated S1 to S6 flanked by amino and carboxyl domains of variable lengths that are thought to be intracellular (Figure below) (Pongs, 1992; Chandy and Gutman, 1995; Jan and Jan, 1997; Coetzee et al., 1999).
Although EAG proteins share with Kv subunits all the main features of proteins of the S4-superfamily, the sequence similarity between EAG and Kv proteins is only ~20% within the hydrophobic core or membrane portion of the proteins, suggesting the two families are only distantly related. Divergence between EAG and Kv families is greatest within the predicted amino and carboxyl termini. In EAG proteins, these regions include novel features not found in Kv or other K+ channel pore-forming subunits and have become defining characteristics of the EAG family. The most striking of these features is the presence of a putative cyclic nucleotide-binding domain (cNBD) in the C-terminus of the protein which resembles that found in cyclic nucleotide-gated channels.

In addition, and unlike all other K+ channel pore-forming subunits, EAG family members share a region of high similarity at the N-terminus of the protein which is critical for the proper functioning of the channels. Recently this domain (residues 1-134), referred to as the "EAG domain", was crystallized from one EAG-family member (human erg1, HERG, Morais et al. 1998). The crystal structure revealed the EAG domain as a member of the PAS domain family. PAS domains are thought to be involved in ligand as well as in protein-protein interactions (Hahn et al., 1997). The structural data of the PAS domain in HERG, combined with mutational analysis of this domain in erg1 and eag1, has provided evidence that this N-terminal domain interacts with the body of the channel, perhaps with residues in the S4-S5 linker, affecting many aspects of channel gating, inactivation and voltage sensitivity (Terlau et al., 1997; Morais et al, 1998; Chen et al, 1999). Sequence diversity within members of the EAG family is concentrated to the C-terminal region following the cNBD.