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. 1998 Sep 15;511(Pt 3):675–682. doi: 10.1111/j.1469-7793.1998.675bg.x

Cloning of a mammalian elk potassium channel gene and EAG mRNA distribution in rat sympathetic ganglia

Wenmei Shi 1, Hong-Sheng Wang 1, Zongming Pan 1, Randy S Wymore 1, Ira S Cohen 1, David McKinnon 1, Jane E Dixon 1
PMCID: PMC2231163  PMID: 9714851

Abstract

  1. Three new members of the EAG potassium channel gene family were identified in rat and the complete coding sequence of one of these genes (elk1) was determined by cDNA cloning.

  2. The elk1 gene, when expressed in Xenopus oocytes, encodes a slowly activating and slowly deactivating potassium channel.

  3. The elk1 gene is expressed in sympathetic ganglia and is also expressed in sciatic nerve.

  4. Six of the seven known EAG genes were found to be expressed in rat sympathetic ganglia, suggesting an important functional role for these channels in the sympathetic nervous system.

A family of three related voltage-gated potassium channel genes, which are distinct from the Shaker class of potassium channel genes, have been previously identified in either Drosophila or mammals (Warmke & Ganetzky, 1994). The first gene identified in this family was the ether àgo-go gene (eag), which was identified initially in Drosophila (Warmke et al. 1991). Two related genes were subsequently described: the eag-related gene (erg) and the eag-like K+ channel gene (elk) (Warmke & Ganetzky, 1994). This family of channel genes is known as the EAG family (Wei et al. 1996). Mammalian homologues of the eag and erg genes have been identified and the potassium channels that they encode have been expressed and characterized (Warmke & Ganetzky, 1994; Ludwig et al. 1994; Robertson et al. 1996; Shi et al. 1997). To date, however, no mammalian homologue of the Drosophilaelk gene has been described, nor have the biophysical properties of any elk channel been characterized. In this paper, three new members of the mammalian EAG gene family are identified. For one of these genes, elk1, we describe the complete coding sequence and have determined the biophysical and pharmacological properties of the encoded channel. In addition, we have determined the mRNA distribution pattern in sympathetic ganglia and sciatic nerve of all known EAG genes.

METHODS

Isolation of cDNA clones

A systematic search for new members of the EAG gene family was conducted using two different methods: (i) novel genes were isolated using degenerate primers combined with polymerase chain reaction (PCR) amplification of cDNAs, and (ii) novel genes were identified in searches of GenBank and cDNAs then isolated by PCR.

Three new genes were identified.

elk1

The elk1 gene was initially identified using the following degenerate primers directed against the S1 and S5 regions to PCR amplify partial cDNA clones from rat brain and superior cervical ganglia (SCG) cDNA.

Forward: TTY AAR RCN RYN TGG GAY TGG.

Reverse: RTA CCA DAT RCA NGC NAG CCA RTG.

An initial sequence encompassing the entire open reading frame of the elk1 gene was determined by performing 5′ and 3′ RACE (rapid amplification of cDNA ends) PCR (Frohman, 1994) using initial anchor oligonucleotides complementary to the partial elk1 cDNA clone. Obtaining a sequence encompassing a complete open reading frame required several rounds of RACE in both directions using SCG cDNA as a template for amplification.

Once cDNAs were obtained that extended beyond both the 5′ and 3′ ends of the open reading frame, oligonucleotides complementary to non-coding regions at either end of the coding sequence were designed. Multiple full-length cDNA clones were amplified in independent PCR reactions from rat SCG cDNA using the Expand High Fidelity PCR system (Boehringer-Mannheim, Indianapolis, IN, USA). The following oligonucleotides were used to amplify full-length cDNA clones, giving a 52 and 381 bp 5′ and 3′ UTR (untranslated regions), respectively.

Forward: CGGGATCCTTGTGGACAAAC.

Reverse: TTCAGGAATGACAACCAGGC.

Two independent clones were sequenced, in their entirety, using a combination of manual and automatic sequencing. Differences between the two sequences were resolved by partial sequencing of a third independent full-length cDNA clone. Sequence alignment of the deduced amino acid sequence was performed using the ClustalW program (Thompson et al. 1994).

elk2

The elk2 gene was initially identified in a search of GenBank using the elk1 sequence as a probe. Two overlapping expressed sequence tags were found to encode a closely related channel (GenBank accession numbers R35526 and R73353). These fragments encompassed part of the cytoplasmic amino terminal domain through to the S2 region. The following complementary oligonucleotide sequences derived from these sequences were used to amplify cDNA clones from rat brain and SCG cDNA.

Forward: GTG ATA CCC ATA AAG AAT GAG.

Reverse: CGG AAA TTC AGC ACA ATG TC.

The elk2 deduced amino acid sequence is: ‘KGEVALFLVSHKDISETKNRGGPDNWKERGGGRRRYGRAGSKGFNANRRRSRAVLYHLSGHLQKQPKGKHKLNKGVFGEKPNLPEYKVAAIRKSPFILLHCGALRATWDGFILLATLYVAVTVPYSVCVSTAREPSAARGPPSVCDLAVEVLFI’.

The elk2 gene was clearly a member of a subfamily of mammalian elk genes. The elk2 deduced amino acid sequence was 52 % identical to elk1 and only 27 and 29 % identical to rat erg1 and eag1, respectively, over the region shown above.

eag2

The eag2 gene was initially identified by PCR amplification from rat brain cDNA using the same primers as were used to isolate the partial rat elk1 cDNA. The reported sequence encompasses a region between S1 and S5. The eag2 deduced amino acid sequence is: ‘VILILTFYTAIMVPYNVSFKTKQNNIAWLVLDSVVDVIFLVDIVLNFHTTFVGPGGEVISDPKLIRMNYLKTWFVIDLLSCLPYDIINAFENVDEGISSLFSSLKVVRLLRLGRVARKLDHYLEYGAAVLVLLVCVFGLVA’.

The eag2 gene was very similar to the previously identified rat eag gene (Ludwig et al. 1994), being 72 % identical to rat eag (eag1) at the DNA sequence level over this region. The deduced amino acid sequence of eag2 was 92 % identical to eag1 over this region and only 42 and 45 % identical to rat elk1 and erg1, respectively.

RNase protection assay

The procedures for the preparation of RNA from sympathetic ganglia and brain and the performance of the RNase protection assay were identical to those described previously (Dixon & McKinnon, 1996). Rats were anaesthetized with sodium pentobarbitone (40 mg kg−1i.p.) and then decapitated before the ganglia were removed. Sciatic nerve RNA was prepared similarly following dissection of sciatic nerve from rat hindlimb and rapid freezing in liquid N2. RNA expression was quantified directly from dried RNase protection gels using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA).

The following DNA templates were used for the RNase protection assays: eag1, which encompasses nucleotides 855 to 1162 of rat eag (Ludwig et al. 1994; accession number Z34264); eag2, which used partial eag2 cDNA described above (accession number AF073891); elk1, which encompasses nucleotides 710 to 1177 of rat elk1 described above (accession number AF061957); elk2, which used partial elk2 cDNA described above (accession number AF073892).

Expression in Xenopus oocytes

Full-length rat elk1 and eag1 cRNA transcripts were synthesized in vitro. Isolation of the full-length rat elk1 cDNA is described above. The full-length rat eag1 cDNA was amplified from rat SCG cDNA using the Expand High Fidelity PCR system. The following oligonucleotides were used to amplify full-length eag1 cDNA clones:

Forward: TGCTGCGGTGAGACACG.

Reverse: TGGTCATGTGTTTGGTGCG.

Oocytes were prepared from mature female Xenopus laevis using established procedures (Colman, 1984). Frogs were anaesthetized in iced water containing a 0.1 % solution of tricaine. Defolliculation was performed by incubation for 2 h in 2 mg ml−1 collagenase (Type VIII, Sigma) in Ca2+-free OR2 oocyte medium (composition (mM): 80 NaCl, 2 KCl, 1 MgCl2, 5 Na-Hepes; pH 7.7) with gentle agitation. Oocytes were stored in OR3 solution (50 % L-15 medium (Gibco), 1 mM glutamine, 15 mM Na-Hepes (pH 7.6), 0.1 mg ml−1 gentamicin) at 18°C. Oocytes were injected with 50 nl of cRNA (∼0.3 ng nl−1) using a microdispenser and a micropipette with a tip diameter of 10–20 μm. Injected oocytes were incubated at 18°C for 24–48 h prior to analysis. After extraction of oocytes, frogs were sutured and monitored for signs of infection in a separate holding tank. No frog developed any infection in the surgical wound.

Oocytes were voltage clamped using a two-microelectrode voltage clamp. Intracellular electrodes filled with 3 M KCl with resistances of 0.5-3 MΩ were used. The standard extracellular recording solution contained (mM): 80 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2 and 5 Na-Hepes (pH 7.7). Data collection and analysis were performed using pCLAMP software (Axon Instruments). E4031 was obtained from Eisai Co. Ltd (Tokodai, Japan).

All animal procedures were approved by the Institutional Animal Care and Use Committee of SUNY at Stony Brook.

RESULTS

Isolation of full-length elk1 cDNA clone

A systematic search for genes related to the Drosophilaelk gene and the mammalian eag gene was conducted and three new genes were identified in rat: elk1, elk2 and eag2 (see Methods). Of these three genes the elk1 gene was of the most interest since no elk channel had been expressed and characterized previously. In addition the elk1 gene is expressed in sympathetic ganglia, a system that has been well characterized both in terms of potassium channel function as well as gene expression (Dixon & McKinnon, 1996; Shi et al. 1997).

The deduced amino acid sequence of the elk1 gene suggests that it encodes a protein 1102 amino acid residues in length with an estimated molecular mass of 123 kDa (Fig. 1). Assignment of the initiator methionine and the start of the open reading frame was facilitated by the high degree of similarity in this region between the elk1 and Drosophilaelk sequences. There were multiple stop codons in all three reading frames around the selected stop codon.

Figure 1. Alignment of the elk, erg and eag deduced amino acid sequences.

Figure 1

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Residues that are identical in two or three sequences are shown with black shading, similar residues are shown with light grey shading and non-conserved residues are shown without shading. The six hydrophobic domains (S1-6), the pore (P) and the putative cyclic nucleotide binding domain (cNBD) are overlined. All sequences are from rat: elk1 (accession number AF061957), erg1 (accession number Z96106) and eag1 (Ludwig et al. 1994; accession number Z34264).

The deduced elk1 amino acid sequence was 41 % identical to the Drosophila elk sequence. Identity with other members of the EAG gene family was significantly lower, ranging from 26 to 31 %. This result suggests that the elk1 gene is a mammalian equivalent of the Drosophilaelk gene. A similar level of identity between mammalian erg genes and their Drosophila counterpart has been found previously (range, 41–44 %; Shi et al. 1997). Identity between the rat eag1 gene and the Drosophilaeag gene is somewhat higher, however, at 51 %.

There is a high degree of similarity between the elk1 deduced amino acid sequence and the erg and eag sequences in the amino terminus, the hydrophobic core and the putative cyclic nucleotide binding domain (Fig. 1). The elk1 sequence shares the pore signature sequence GFG with both the erg and eag channels, in contrast to most other potassium channels, which have GYG in the equivalent position (Wei et al. 1996). The elk1 protein has a relatively large cytoplasmic carboxyl domain compared with the other two channels and there is a distinctive run of negatively charged residues in this region, with eleven of twelve residues being either glutamate or aspartate. A similar but much shorter sequence is seen in eag1 in the equivalent position.

Functional expression of elk1 channels

It was of interest to compare the biophysical properties of the elk1 channel with those of eag and erg channels, since all three channels share significant regions of sequence identity. Eag and erg channels have one common feature: they are relatively slowly activating compared with Kv class (Shaker-related) channels. This property is also shared by the elk1 channel (Fig. 2A). The elk1 channel is one of the slowest activating EAG channels, having a time constant for activation of 676 ± 37 ms (n = 7) at 0 mV. The threshold for activation is around -40 mV and the conductance-voltage curve can be fitted with a simple Boltzmann distribution (Fig. 2B: mid-point for activation, Vn= 9.3 ± 0.4 mV and slope factor, kn= -13.1 ± 0.3 mV (n = 7)).

Figure 2. Properties of the rat elk1 channel expressed in Xenopus oocytes.

Figure 2

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A, voltage-clamp recordings of elk1 currents. Recordings show current responses to voltage steps over the range -60 to +40 mV in 10 mV increments, from a holding potential of -70 mV. Tail currents were recorded at -50 mV. B, normalized conductance-voltage curve of the elk1 channel. Conductance of the channel was calculated as the current measured at the end of a 2 s depolarizing step divided by the driving force (equilibrium potential for K+, EK= -75 mV). Data points are averages from 7 oocytes and were fitted with the equation:
graphic file with name tjp0511-0675-mu1.jpg
where Vn, the mid-point for activation, was 9.3 ± 0.4 mV and kn, the slope factor, was -13.1 ± 0.3 mV. Error bars are s.e.m. and are not as large as the graph symbols. C, activation of elk1 and rat eag1 channels from different holding potentials. Holding potentials were varied over the range -120 to -40 mV in 20 mV increments. Test potential was 0 mV. Note the difference in time scale for the two types of channel. D, deactivation of elk1 tail currents. Currents were activated by a 2500 ms step to +10 mV followed by steps to potentials over the range -90 to -30 mV in 10 mV increments. Time constants for deactivation are indicated next to the appropriate trace. The trace at -70 mV is not shown due to the small amplitude of the tail current. E, activation of the elk1 channel in control perfusion solution (pH = 7.7, left) and in perfusion solution with pH lowered to 6.6 (right). Recordings show current responses to voltage steps from a holding potential of -70 mV. Test potentials are indicated next to selected current traces. F, conductance- voltage curves in perfusion solutions with various pH values. Data points were fitted with the equation:
graphic file with name tjp0511-0675-mu2.jpg
where the mid-point for activation Vn= 9.6, 8.6, 15.7, 25.2 and 9.0 mV, the slope factor kn= -13.8, -13.7, -12.5, -12.3 and -13.4 mV and the maximum conductance Gmax= 26.8, 26.3, 24.1, 21.8 and 25.8 μS for pH = 7.7, 8.6, 7.1, 6.6 and wash (7.7), respectively. G, E4031 does not block the elk1 channel. Voltage-clamp protocol the same as in A. H, the elk1 channel is blocked by 1 mM Ba2+. Voltage-clamp protocol the same as in A.

In a simple activation protocol such as the one shown in Fig. 2A, the elk1 channel has a similar waveform to eag channels: it is a slowly activating outward rectifier. The elk1 channel exhibits no apparent inactivation during depolarizing voltage steps of up to several seconds, similar to the mammalian eag1 channel (Ludwig et al. 1994; Robertson et al. 1996). In contrast, erg channels have very fast inactivation rates, significantly faster than their activation rates, which results in an apparent inward rectification at positive membrane potentials (Sanguinetti et al. 1995; Spector et al. 1996; Shi et al. 1997).

One particularly striking feature of the eag channels is that the rate of activation is strongly dependent upon the holding potential: activation is significantly faster from depolarized than hyperpolarized holding potentials. This suggests that during the activation process the eag channel undergoes a slow, voltage-dependent transition between at least two distinct closed states before reaching the open state (Ludwig et al. 1994; Robertson et al. 1996). The elk1 channel shows no obvious change in the activation rate from different holding potentials, in marked contrast to the results for eag channels over a similar range of holding potentials (Fig. 2C). In this respect, the elk1 channel is more similar to erg channels, which also do not exhibit a significant shift in activation rates with changes in holding potential.

There are some other kinetic properties for which the elk1 channel is more similar to erg than eag channels. The rate of deactivation of the elk1 channel is relatively slow, with a time constant of 111 ± 7 ms (n = 7) at -50 mV (Fig. 2D). Deactivation rates for erg channels are also very slow, with deactivation time constants in the range of 100 ms up to several seconds at -50 mV (Sanguinetti et al. 1995; Shi et al. 1997). In marked contrast, the deactivation rate of the mammalian eag1 channel is very fast, with a time constant of 1–6 ms (Terlau et al. 1997).

Activation of the elk1 channel was strongly dependent on the pH of the external bath solution (Fig. 2E). Following acidification of the external perfusate solution, the conductance-voltage curve was shifted significantly to more positive membrane potentials (Fig. 2F). The mid-point of the activation curve was shifted from 10.4 ± 0.4 mV at pH = 7.7, to 17.0 ± 0.7 mV at pH = 7.1 (P < 0.05), to 28.1 ± 1.5 mV at pH = 6.6 (P < 0.001). The slope factor was not significantly affected by changes in external pH (P > 0.5). Changing the external solution to a more alkaline pH did not produce significant shifts in the activation- conductance curve (Fig. 2F). A somewhat similar dependence of activation on external pH is seen for mammalian eag channels (Terlau et al. 1996), although for eag channels there is also significant slowing of the activation kinetics by acidic pH, which is not seen for elk1 channels.

The elk1 channel was almost completely resistant to the application of two common potassium channel blockers, TEA and 4-AP. Concentrations of up to 10 mM of either drug had limited or no effects on the channel. Another class of potassium channel blockers, methanesulphonanilide drugs such as E4031, are useful reagents because they are apparently quite selective for cloned and native erg channels at concentrations less than ∼10 μm (Sanguinetti & Jurkiewicz, 1990; Shi et al. 1997). These compounds have not been tested systematically on closely related channels, however, and for this reason we examined the effects of E4031 on the elk1 channel. E4031 had almost no effect at concentrations up to 10 μm (Fig. 2G). Barium ions also have some potential for differentiating among the various EAG family channels. The elk1 channel is more sensitive than the eag1 channel to Ba2+ ions, being 85 % blocked by 1 mM Ba2+ in the external solution (Fig. 2H). In comparison, the eag1 channel is blocked less than 40 % by 1 mM Ba2+ (see also Terlau et al. 1996).

EAG gene expression in sympathetic ganglia

To date the functional roles of eag, erg and elk channels have been poorly characterized in vivo. One potentially useful model system to study the function of these channels is the peripheral sympathetic ganglia. For this reason, we determined the distribution of elk and eag gene expression in three sympathetic ganglia: one paravertebral ganglia, the superior cervical ganglia (SCG) and two prevertebral ganglia, the coeliac ganglia (CG) and the superior mesenteric ganglia (SMG) (Fig. 3A). The elk1 gene is expressed in both prevertebral and paravertebral ganglia at approximately equal levels. In contrast, the elk2 gene is not expressed at detectable levels in sympathetic ganglia, although it is robustly expressed in the brain. Both eag genes are expressed in sympathetic ganglia. The eag1 gene is expressed at high levels in all three sympathetic ganglia. The eag2 gene is only expressed at significant levels in the prevertebral ganglia, the CG and SMG.

Figure 3. EAG potassium channel mRNA expression in sympathetic ganglia, brain and sciatic nerve determined by RNase protection analysis.

Figure 3

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A, samples tested were superior cervical ganglia (SCG), coeliac ganglia (CG), superior mesenteric ganglia (SMG) and brain. For the sympathetic ganglia the sample always contained 5 μg of total RNA. A variable amount of total RNA was included in the brain samples as noted. a, elk1 mRNA was expressed in all three sympathetic ganglia. The brain sample contained 5 μg of total RNA. b, elk2 mRNA was not expressed in sympathetic ganglia but was moderately abundant in brain. The brain sample contained 2.5 μg of total RNA. c, eag1 mRNA was expressed at equal levels in all three ganglia and was also expressed in brain. The brain sample contained 14 μg of total RNA. d, eag2 mRNA was barely detectable in the SCG but was expressed in the two prevertebral ganglia, the CG and SMG, as well as in brain. The brain sample contained 14 μg of total RNA. B, elk1 and erg1 mRNA expression in sciatic nerve (SN). Superior cervical ganglia (SCG) RNA served as a positive control. All samples contained 5 μg of total RNA. The cyclophilin gene (cyc) was used as an internal positive control in all experiments to check for sample loss and equal loading.

In addition to neurones, sympathetic ganglia also contain glial cells (known as satellite cells and developmentally related to Schwann cells). To determine whether gene expression in ganglia could be due to expression in glial cells we examined EAG gene expression in sciatic nerve, which contains a relatively pure population of Schwann cells. Two of the seven EAG genes tested are expressed in sciatic nerve, elk1 and erg1 (Fig. 3B).

A summary of EAG gene expression in sympathetic ganglia and sciatic nerve is shown in Table 1. At least six EAG genes are expressed in the sympathetic ganglia and all seven EAG genes are expressed in at least one neuronal tissue. Two genes, eag2 and erg2, are differentially expressed in sympathetic ganglia. Both genes are expressed at higher levels in prevertebral ganglia than paravertebral ganglia. The housekeeping gene cyclophilin was expressed at significantly lower levels in sciatic nerve RNA than in ganglia or brain RNA (Fig. 3B). It is possible, therefore, that mRNA levels, as a fraction of total RNA, are significantly lower in sciatic nerve than in ganglia or brain. For this reason, the relative level of elk1 and erg1 expression in sciatic nerve described in Table 1 may be an underestimate in comparisons between sciatic nerve and ganglia or brain.

Table 1.

Distribution of EAG potassium channel gene expression in rat sympathetic ganglia, brain and sciatic nerve

SCG CG SMG Brain Sciatic nerve
eag1 +++ +++ +++ ++++ —
eag2 +/− + + ++ —
erg1 +++++ +++++ +++++ +++++ +++
erg2 + ++ +++ — —
erg3 +++ +++ +++ ++++ —
elk1 +++ +++ +++ +/− ++
elk2 — — — +++ —
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+++++, very high; ++++, high; +++, moderate; ++, low; +, very low; +/−, just detectable; -, negative. Semi-quantitative analysis of the relative levels of expression of all the EAG genes was achieved by a combination of radioactive counting of individual gels and visual comparison of autoradiographs from multiple experiments. In every case the data are based on multiple independent experiments (n = 2 to 6). Data for sciatic nerve may be subject to a systematic underestimate (see Results). Data for the erg gene expression in sympathetic ganglia is adapted from Shi et al. (1997).

DISCUSSION

In this paper we describe the complete coding sequence of a new potassium channel gene, the rat elk1 gene, and partial sequences for two other EAG genes, eag2 and elk2. The elk1 and elk2 genes are mammalian equivalents of the Drosophilaelk gene (Warmke & Ganetzky, 1994). When expressed in a heterologous expression system, the elk1 channel has some similar biophysical properties compared with the related eag and erg channels, but also has some clearly distinct properties. In particular, the elk1 channel activates very slowly and also deactivates slowly. The elk1 channel shows little or no inactivation during sustained depolarizing voltage steps.

Identification of three new EAG genes in addition to the previously known genes and the demonstration that all the EAG genes are expressed in at least some region of the nervous system raises obvious questions about the function of EAG channels in the mammalian nervous system. At least six EAG genes are expressed in sympathetic ganglia, which is a large number of related genes to be expressed in a tissue that contains a relatively homogeneous population of cells. This result suggests that EAG channels are functionally important with, at most, only partially overlapping functions among the different family members. To date, however, there is little convincing data on the physiological function of any of these channels in the mammalian nervous system. In Drosophila, mutations in the eag and erg genes produce hyperexcitability, suggesting a role for these channels in regulating neuronal electrical activity (Ganetzky & Wu, 1983; Titus et al. 1997; Wang et al. 1997). The specific cause of hyperexcitability and even whether these channels are neuronal in origin has yet to be definitively established.

Very little information is currently available on the physiological role of elk channels or the related eag channels in mammals. Both channels have relatively positive activation curves which limit their ability to affect sub-threshold electrical excitability. They activate too slowly to affect the shape of the action potential and, even if they are neuronally expressed, it is difficult to understand how they might affect neuronal excitability. It is possible that heteromultimerization with β-subunits might modify the functional properties of these channels but there is currently no direct evidence to support such a possibility. The elk1 gene is expressed in sciatic nerve, which suggests that elk1 channels may function in the glial cells of sympathetic ganglia rather than in neurones.

It has been suggested that eag subunits co-assemble with more rapidly activating Kv channel subunits to produce heteromers with novel kinetic properties. The Drosophila eag channel appears to modify the inactivation properties of the Shaker B channel, possibly by co-assembly into a heteromeric channel (Chen et al. 1996). It has yet to be shown, however, that the mammalian Kv channels can be modified by co-assembly with mammalian eag subunits. A recent report of a current expressed in human neuroblastoma cells that has striking similarities to homomeric eag channels expressed in heterologous expression systems (Meyer & Heinemann, 1998) suggests that mammalian eag subunits may assemble as independent channels rather than as components of heteromultimeric complexes.

In conclusion, there are a large number of EAG channels expressed in the mammalian nervous system and these channels have a wide diversity of biophysical properties. Surprisingly, the physiological function that any EAG channel performs in the nervous system has yet to be determined. In the past, the sympathetic nervous system has proved to be a good model system in which to study potassium channels and a large number of different potassium channel currents have been identified (Brown et al. 1982; Pennefather et al. 1985; Belluzzi & Sacchi, 1991; Wang & McKinnon, 1995). There is, however, a clear excess in the number of potassium channel genes expressed in sympathetic ganglia relative to the number of physiologically identified potassium currents. It will be technically difficult to refine further the electrophysiological techniques necessary to resolve more distinct potassium currents although single channel recordings have provided anecdotal evidence for diversity within apparently homogeneous macroscopic currents. These technical limitations may make it necessary to use genetic approaches such as gene knock-outs to establish the physiological function of the EAG channels in the mammalian nervous system.

Acknowledgments

This work was supported by grants NS-29755, NS-01718 and HL-20558 from the National Institutes of Health.

References

  1. Belluzzi O, Sacchi O. A five conductance model of the action potential in the rat sympathetic neurone. Progress in Biophysics and Molecular Biology. 1991;55:1–30. doi: 10.1016/0079-6107(91)90009-h. [DOI] [PubMed] [Google Scholar]
  2. Brown DA, Adams PR, Constanti A. Voltage-sensitive K-currents in sympathetic neurons and their modulation by neurotransmitters. Journal of the Autonomic Nervous System. 1982;6:23–35. doi: 10.1016/0165-1838(82)90019-4. [DOI] [PubMed] [Google Scholar]
  3. Chen ML, Hoshi T, Wu CF. Heteromultimeric interactions among K+ channel subunits from Shaker and eag families in Xenopus oocytes. Neuron. 1996;17:535–542. doi: 10.1016/s0896-6273(00)80185-3. [DOI] [PubMed] [Google Scholar]
  4. Colman A. Translation of eukaryotic messenger RNA in Xenopus oocytes. In: Hames BD, Higgins SJ, editors. Transcription and Translation. Oxford: IRL Press; 1984. pp. 271–302. [Google Scholar]
  5. Dixon JE, McKinnon D. Potassium channel mRNA expression in prevertebral and paravertebral sympathetic neurons. European Journal of Neuroscience. 1996;8:183–191. doi: 10.1111/j.1460-9568.1996.tb01179.x. [DOI] [PubMed] [Google Scholar]
  6. Frohman MA. On beyond classic RACE (rapid amplification of cDNA ends) PCR Methods and Applications. 1994;4:S40–58. doi: 10.1101/gr.4.1.s40. [DOI] [PubMed] [Google Scholar]
  7. Ganetzky B, Wu CF. Neurogenetic analysis of potassium currents in Drosophila: synergistic effects on neuromuscular transmission in double mutants. Journal of Neurogenetics. 1983;1:17–28. doi: 10.3109/01677068309107069. [DOI] [PubMed] [Google Scholar]
  8. Ludwig J, Terlau H, Wunder F, Bruggemann A, Pardo LA, Marquardt A, Stuhmer W, Pongs O. Functional expression of a rat homologue of the voltage gated ether a go-go potassium channel reveals differences in selectivity and activation kinetics between the Drosophila channel and its mammalian counterpart. EMBO Journal. 1994;13:4451–4458. doi: 10.1002/j.1460-2075.1994.tb06767.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Meyer R, Heinemann SH. Characterization of an eag-like potassium channel in human neuroblastoma cells. The Journal of Physiology. 1998;508:49–56. doi: 10.1111/j.1469-7793.1998.049br.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Pennefather P, Lancaster B, Adams PR, Nicoll RA. Two distinct Ca-dependent K currents in bullfrog sympathetic ganglion cells. Proceedings of the National Academy of Sciences of the USA. 1985;82:3040–3044. doi: 10.1073/pnas.82.9.3040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Robertson GA, Warmke JW, Ganetzky B. Potassium currents expressed from Drosophila and mouse eag cDNAs in Xenopus oocytes. Neuropharmacology. 1996;35:841–850. doi: 10.1016/0028-3908(96)00113-x. 10.1016/0028-3908(96)00113-X. [DOI] [PubMed] [Google Scholar]
  12. Sanguinetti MC, Jiang C, Curran ME, Keating MT. A mechanistic link between an inherited and an acquired cardiac arrhythmia: HERG encodes the IKr potassium channel. Cell. 1995;81:299–307. doi: 10.1016/0092-8674(95)90340-2. [DOI] [PubMed] [Google Scholar]
  13. Sanguinetti MC, Jurkiewicz NK. Two components of cardiac delayed rectifier K+ current: differential sensitivity to block by class III antiarrhythmic agents. Journal of General Physiology. 1990;96:195–215. doi: 10.1085/jgp.96.1.195. 10.1085/jgp.96.1.195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shi W, Wymore RS, Wang H-S, Pan Z, Cohen IS, Mckinnon D, Dixon JE. Identification of two nervous system specific members of the erg potassium channel gene family. Journal of Neuroscience. 1997;17:9423–9432. doi: 10.1523/JNEUROSCI.17-24-09423.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Spector PS, Curran ME, Zou A, Keating MT, Sanguinetti MC. Fast inactivation causes rectification of the IKr channel. Journal of General Physiology. 1996;107:611–619. doi: 10.1085/jgp.107.5.611. 10.1085/jgp.107.5.611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Terlau H, Heinemann SH, Stuhmer W, Pongs O, Ludwig J. Amino terminal-dependent gating of the potassium channel rat eag is compensated by a mutation in the S4 segment. The Journal of Physiology. 1997;502:537–543. doi: 10.1111/j.1469-7793.1997.537bj.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Terlau H, Ludwig J, Steffan R, Pongs O, Stuhmer W, Heinemann SH. Extracellular Mg2+ regulates activation of rat eag potassium channel. Pflügers Archiv. 1996;432:301–312. doi: 10.1007/s004240050137. [DOI] [PubMed] [Google Scholar]
  18. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Research. 1994;22:4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Titus SA, Warmke JW, Ganetzky B. The Drosophilaerg K+ channel polypeptide is encoded by the seizure locus. Journal of Neuroscience. 1997;17:875–881. doi: 10.1523/JNEUROSCI.17-03-00875.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Wang H-S, Mckinnon D. Potassium channel expression in prevertebral and paravertebral sympathetic neurones: control of firing properties. The Journal of Physiology. 1995;485:319–335. doi: 10.1113/jphysiol.1995.sp020732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wang X, Reynolds ER, Deak P, Hall LM. The seizure locus encodes the Drosophila homolog of the HERG potassium channel. Journal of Neuroscience. 1997;17:882–890. doi: 10.1523/JNEUROSCI.17-03-00882.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Warmke J, Drysdale R, Ganetzky B. A distinct potassium channel polypeptide encoded by the Drosophilaeag locus. Science. 1991;252:1560–1562. doi: 10.1126/science.1840699. [DOI] [PubMed] [Google Scholar]
  23. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proceedings of the National Academy of Sciences of the USA. 1994;91:3438–3442. doi: 10.1073/pnas.91.8.3438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wei A, Jegla T, Salkoff L. Eight potassium channel families revealed by the C. elegans genome project. Neuropharmacology. 1996;35:805–829. doi: 10.1016/0028-3908(96)00126-8. [DOI] [PubMed] [Google Scholar]

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