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Volume 17, Number 24,
Issue of December 15, 1997
Identification of Two Nervous System-Specific Members of the
erg Potassium Channel Gene Family
Wenmei Shi1,
Randy S. Wymore2,
Hong-Sheng Wang2,
Zongming Pan1,
Ira S. Cohen1,
David McKinnon1, 2, and
Jane E. Dixon1
1 Departments of Neurobiology and Behavior and
2 Physiology and Biophysics, State University of New York at
Stony Brook, Stony Brook, New York 11794
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Two new potassium channel genes, erg2 and
erg3, that are expressed in the nervous system of the
rat were identified. These two genes form a small gene family with the
previously described erg1 (HERG) gene. The
erg2 and erg3 genes are expressed
exclusively in the nervous system, in marked contrast to
erg1, which is expressed in both neural and non-neural
tissues. All three genes are expressed in peripheral sympathetic
ganglia. The erg3 channel produces a current that has a
large transient component at positive potentials, whereas the other two
channels are slowly activating delayed rectifiers. Expression of the
erg1 gene in the sympathetic nervous system has
potential implications for the etiology of the LQT2 form of the human
genetic disease long QT syndrome.
Key words:
potassium channel;
erg;
long QT syndrome;
sympathetic neuron;
gene;
delayed rectifier
INTRODUCTION
A family of three related
voltage-gated potassium channel genes (eag, erg,
and elk) has been described in either Drosophila or mammals (Warmke et al., 1991
; Ludwig et al., 1994; Warmke and Ganetzky, 1994; Titus et al., 1997; Wang et al., 1997). These channels
share the six-membrane-spanning architecture of the Kv class
(Shaker-related) of voltage-gated potassium channels but otherwise are distantly related to the Kv class channels (Warmke and
Ganetzky, 1994). The channels encoded by the eag-related
genes are relatively slowly activating, as compared with Kv class
potassium channels (Ludwig et al., 1994; Sanguinetti et al., 1995;
Terlau et al., 1996), and have some similarities to slowly activating potassium currents that are important in determining the threshold firing properties of neurons (Brown, 1988; Yamada et al., 1989; Wang
and McKinnon, 1995). As might be anticipated from the biophysical properties of these channels, mutations in either the eag
gene or the erg gene of Drosophila result in a
hyperexcitable phenotype (Ganetzky and Wu, 1983; Titus et al., 1997;
Wang et al., 1997).
The erg gene recently has become the center of considerable
interest because mutations in this gene have been shown to underlie one
form of a human genetic disease known as long QT syndrome, which gives
rise to arrhythmias and an increased incidence of sudden death (Curran
et al., 1995; Sanguinetti et al., 1996a). It is thought that the
erg gene encodes the channel that underlies a previously
identified potassium current known as IKr
(Sanguinetti et al., 1995, 1996a; Trudeau et al., 1995). This current
was described originally in cardiac myocytes (Shibasaki, 1987;
Sanguinetti and Jurkiewicz, 1990), and it has been suggested that
mutations in the erg gene may increase the susceptibility to
arrhythmia, possibly by causing a prolongation of the cardiac action
potential (Curran et al., 1995; Sanguinetti et al., 1996a).
Alternatively, it had been suggested previously that dysfunction in the
control of sympathetic outflow to the heart can induce the
life-threatening arrhythmias that are characteristic of long QT
syndrome (Schwartz et al., 1994).
We have shown that transcripts from the erg gene are
expressed abundantly in the rat and human nervous systems, suggesting that the erg gene product might contribute to nervous system
function (Wymore et al., 1997). The observation that mutations in the
erg gene of Drosophila produce clear neurological
defects adds further weight to this possibility (Titus et al., 1997;
Wang et al., 1997). To examine further the role of the erg
gene in the mammalian nervous system, we have conducted a systematic
screen to identify genes that are related to erg. We have
concentrated particularly on the sympathetic nervous system because of
the potential clinical importance of erg channels in this
tissue. In this paper we describe two new members of the erg
gene family (erg2 and erg3) that are expressed
exclusively in the nervous system and are expressed abundantly in
sympathetic ganglia.
MATERIALS AND METHODS
Isolation of partial cDNA clones. Two sets of
degenerate oligonucleotides directed against conserved regions of the
eag, erg, and elk gene family were
designed.
Set 1 was directed against the amino acid sequences "APQNTF" and
"FK(AT)(ITV)WDW": forward, GC(ACGT) CC(ACGT) CA(AG) AA(CT) AC(ACGT)
TT(CT); and reverse, CCA (AG)TC CCA (ACGT)(AG)(CT) (ACGT)G(CT) (CT)TT
(AG)AA.
Set 2 was directed against the amino acid sequences
"FK(AT)(ITV)WDW" and "HWLACIWY": forward, TT(CT) AA(AG)
(AG)C(ACGT) (AG)(CT)(ACGT) TGG GA(CT) TGG; and reverse,
(AG)TA CCA (AGT)AT (AG)CA (ACGT)GC (ACGT)AG CCA (AG)TG.
Using these two sets of oligonucleotides, we prepared cDNA
fragments by reverse transcription and PCR amplification from total cellular RNA isolated from rat superior cervical ganglia (SCG). The
amplified cDNA fragments were gel-purified and then subcloned into
pBluescript SK II (Stratagene, La Jolla, CA) and analyzed by manual
sequencing. Using this procedure, we identified two novel classes of
erg-related cDNA clones. The erg3 cDNA initially was isolated by using the first set of oligonucleotides, and the erg2 cDNA was identified by using the second set.
Isolation of full-length ERG2 and ERG3 cDNA clones.
Full-length rat erg2 and erg3 cDNA sequences
were obtained first by performing a modified 5
and 3 rapid
amplification of cDNA ends (RACE) protocol (essentially as described by
Frohman, 1994), using anchor oligonucleotides complementary to the
partial erg2 and erg3 clones. This required several rounds of RACE in both directions to obtain sequences with
complete open reading frames. For erg2 the tissue source for
synthesizing the cDNA used in the RACE protocol was celiac ganglia, and
for erg3 the tissue source was brain.
Once cDNAs were obtained that extended beyond both the 3 and 5 ends
of the open reading frame, oligonucleotides complementary to noncoding
regions at either end of the coding sequence were designed, and
full-length cDNA clones were obtained by using the Expand High Fidelity
PCR system (Boehringer Mannheim, Indianapolis, IN) for PCR
amplification. For erg2, the following oligonucleotides were
used to amplify full-length cDNA clones from rat celiac ganglia RNA,
giving a 32 and 497 bp 5 and 3 untranslated region (UTR), respectively: forward, GAG TAA CTC CCA GCA AGT GC; and reverse, ACT GTT
ATG AGA GTC TCA GGG G.
For erg3, the following oligonucleotides were used to
amplify full-length cDNA clones from rat brain RNA, giving a 182 and 37 bp 5 and 3 UTR, respectively: forward, GAT GGA TTG GAC TTC GGC; and
reverse, GCA CTT ACA TTG GAT GTG GAG.
Full-length cDNAs were subcloned into the pBluescript SK II vector. Two
erg2 clones were sequenced in their entirety, using a
combination of manual and automatic sequencing. One erg3
clone was sequenced in its entirety, using a combination of manual and automatic sequencing; a second independent sequence was obtained by
sequencing the erg3 clones obtained by RACE. Differences
between the two sequences were resolved by partial sequencing of a
second full-length erg3 cDNA clone, which also was obtained
from brain. Sequence alignment was performed with the Clustal W program
(Thompson et al., 1994). Sequences were submitted to GenBank, with
accession numbers AF016192 and AF016191 for erg2 and
erg3, respectively.
Preparation of RNA. Tissue samples were quick-frozen in
liquid N2 and then homogenized in guanidinium thiocyanate.
Total RNA was prepared by pelleting the homogenate over a CsCl step
gradient as described previously (Dixon and McKinnon, 1996).
Poly(A+) RNA was prepared with paramagnetic poly-dT
beads (Dynal, Oslo, Norway) as described previously (Wymore et al.,
1997). All RNA samples were quantitated carefully by spectrophotometric
analysis.
RNase protection assay. RNA probes were prepared as
described previously (Dixon and McKinnon, 1994). In all cases a
significant amount of nonhybridizing sequence (~50-80 bp) was
included in the probe to allow for easy distinction between the probe
and the specific protected band. The specificity of the assay was such
that there was no evidence for unwanted cross-reaction between any
probe and another nonspecific potassium channel transcript.
RNase protection assays were performed as described previously (Dixon
and McKinnon, 1996). For each sample point 5 µg of total RNA or 1 µg of poly(A+) RNA was used in the assay. A
species-specific cyclophilin probe was included in the hybridization as
an internal control to confirm that the sample was not lost or degraded
during the assay. Five micrograms of yeast tRNA were used as a negative
control to test for the presence of probe self-protection bands. RNA
expression was quantitated directly from dried RNase protection gels
with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Expression of erg channels in Xenopus
oocytes. Full-length erg1, erg2, and
erg3 cRNA transcripts were synthesized in vitro. The erg1 clone was a human cDNA that has been described
previously (Sanguinetti et al., 1995) (a gift from Dr. Michael
Sanguinetti, University of Utah).
Oocytes were prepared from mature female Xenopus laevis,
using established procedures (Colman, 1984). Defolliculation was performed by incubation for 2 hr in 2 mg/ml collagenase (Type VIII,
Sigma, St. Louis, MO) in Ca2+-free OR2 oocyte medium
with gentle agitation. Oocytes were stored in OR3 solution [50% L-15
medium (Life Technologies, Gaithersburg, MD), 1 mM
glutamine, 15 mM Na-HEPES, pH 7.6, and 0.1 mg/ml
gentamicin] at 18°C. Oocytes were injected with 50 nl of cRNA
(~0.3 ng/nl) by using a microdispenser and a micropipette with a tip
diameter of 10-20 µm. Injected oocytes were incubated at 18°C for
24-48 hr before analysis.
Oocytes were voltage-clamped with a two-microelectrode voltage clamp.
Intracellular electrodes filled with 3 M KCl with
resistances of 0.5-3 M
were used. The standard extracellular
recording solution contained (in mM): 80 NaCl, 5 KCl, 1.8 CaCl2, 1 MgCl2, and 5 Na-HEPES, pH 7.6. Data collection and analysis were performed with pClamp software (Axon Instruments, Foster City, CA). The methanesulfonanilide E4031 was obtained from Eisai Company (Tokodai, Japan).
RESULTS
Isolation and properties of the erg2 and
erg3 cDNA clones
Two partial cDNA clones, which identified two novel potassium
channel genes, were isolated from rat SCG cDNA. The cDNAs were cloned
by PCR, with primers that were directed against conserved regions of
the eag, erg, and elk gene families.
These partial sequences were closely related to the previously
described h-erg (or HERG) gene (Warmke and Ganetzky, 1994),
which we will refer to in the following discussions as erg1.
Full-length erg2 and erg3 cDNA clones were
obtained (see Materials and Methods for details) and sequenced in their
entirety (Fig. 1). Identification of the
initiator methionine and the start of the open reading frame was
facilitated by the high degree of similarity in this region among the
three genes. There is a short region of conservation in the three
sequences at the C terminus immediately before the presumptive stop
codon, which suggests that the derived amino acid sequences correctly
identify the entire coding regions of all three genes.
Fig. 1.
Alignment of the erg1,
erg2, and erg3 deduced amino acid
sequences. There is 63% identity between erg2 and
erg1, 57% identity between erg3 and
erg1, and 61% identity between erg2 and
erg3. Residues that are identical in all three sequences
are shown with black shading, residues identical in two
sequences are shown with dark gray shading, similar
residues are shown with light gray shading, and
nonconserved residues are shown without shading. The
erg1 sequence corresponds to the human
erg gene (Warmke and Ganetzky, 1994). The
erg2 and erg3 sequences are from rat. The six hydrophobic domains (S1-S6), the pore
(P), and the putative cyclic nucleotide binding
domain (cNBD) are overlined.
[View Larger Version of this Image (144K GIF file)]
At the amino acid level the three mammalian erg genes had
identity scores of ~60% over the entire deduced amino acid sequence (Table 1). The erg2 sequence
was 63% identical and the erg3 sequence was 57% identical
to the erg1 gene, and the erg2 sequence was 61%
identical to the erg3 sequence. All three mammalian
erg sequences had similar identity scores (~42%) in
comparisons with the Drosophila erg gene, suggesting that
they all derive from a common gene. Identity with the other members of
the eag gene family (eag and elk) was
significantly lower, ~30% overall. This result suggests that the
erg1, erg2, and erg3 genes form a
distinct subfamily.
There are three highly conserved domains in the three sequences: the
initial N-terminal sequence, the hydrophobic core, and the putative
cyclic nucleotide binding domain. Curiously, the Drosophila
erg sequence lacks this conserved N-terminal domain (Titus et al.,
1997; Wang et al., 1997), although it is present in all three mammalian
erg sequences as well as in the eag and elk sequences. It is known that the mammalian erg
genes can produce alternative spliced transcripts that lack the
N-terminal coding region (London et al., 1997), and it is possible that
there is also alternative splicing of the Drosophila erg
gene products and that some Drosophila erg mRNA species may
include this domain. A short, completely conserved motif (SDPG) is
found in the extreme C terminus of all three mammalian erg
sequences.
Tissue distribution of erg2 and erg3
gene expression
The erg1 gene is expressed in the hearts of all species
tested to date (Wymore et al., 1997) and is thought to encode one component of the delayed rectifier potassium current found in heart
(Sanguinetti et al., 1995; Trudeau et al., 1995). Mutations in the
erg1 gene have been shown to be associated with the LQT2 form of the human genetic disease long QT syndrome, which involves a
significantly increased susceptibility to arrhythmias triggered by
emotional or physical stress (Curran et al., 1995; Sanguinetti et al.,
1996a). For this reason, we first examined erg2 and
erg3 mRNA expression in heart. Neither gene is expressed at
detectable levels in either atrial or ventricular muscle, in marked
contrast to the results obtained with the erg1 gene (Fig.
2A).
Fig. 2.
erg potassium channel mRNA
expression in heart and neural tissues determined by RNase protection
analysis. A, Neither erg2 nor
erg3 mRNA is expressed at detectable levels in atrial or
ventricular (Vent) muscle, in marked contrast to
erg1, which is abundant in both tissues
(arrows). The positive control samples are celiac ganglia (CG) or brain mRNA. B, All three
erg genes are expressed in neural tissue. Samples tested
were brain, superior cervical ganglia (SCG), celiac
ganglia (CG), superior mesenteric ganglia (SMG), and retina. The cyclophilin gene
(cyc, arrows) was used as an internal
positive control; as has been shown previously, cyclophilin expression
was always lower in muscle tissues, as compared with other
tissues.
[View Larger Version of this Image (65K GIF file)]
As shown previously (Wymore et al., 1997), the erg1 gene is
expressed abundantly in brain and in retina. Intriguingly, given the
clinical symptoms associated with mutations in the erg1
gene, erg1 mRNA is expressed abundantly in sympathetic
ganglia (Fig. 2B). This result suggests that
mutations in the erg1 gene could affect sympathetic
regulation of cardiac function in addition to having direct effects on
myocardial function.
The erg2 gene has a very restricted distribution in neural
tissues and is not expressed at detectable levels in brain. Although initially cloned by PCR amplification from SCG mRNA, erg2
expression in SCG is also very low. The peripheral sympathetic nervous
system has two anatomically and functionally distinct components: the paravertebral ganglia and the prevertebral ganglia. It has been shown
previously that the electrophysiological properties of neurons in the
paravertebral ganglia, such as the SCG, are very uniform, whereas there
are at least two electrophysiologically distinct types of neurons in
the prevertebral ganglia (Cassell et al., 1986; Wang and McKinnon,
1995, 1996). The molecular basis for this differentiation currently is
undetermined (Dixon and McKinnon, 1996). The erg2 gene is
expressed abundantly in the two prevertebral sympathetic ganglia
examined: the celiac ganglia (CG) and the superior mesenteric ganglia
(SMG) (Fig. 2B). This result suggests that the
erg2 channel could contribute to the electrophysiological differentiation of prevertebral neurons, but identification of the
native current corresponding to the erg2 channel is
necessary before this conclusion can be established. There is also a
low level of erg2 expression in retina.
The erg3 gene is broadly expressed throughout the nervous
system, similar to erg1. In addition to brain,
erg3 mRNA is expressed in all of the sympathetic ganglia
tested and also is expressed at low levels in retina (Fig.
2B).
Expression of all three erg genes in sympathetic ganglia
prompted us to examine expression in PC12 cells, a cell line that often
is used as a cell culture analog of sympathetic neurons. Both the
erg1 and erg2 genes are expressed in PC12 cells,
whereas the erg3 gene is not expressed (Fig.
3A). A further elaboration of
the neuronal phenotype of PC12 cells can be induced by treatment with
nerve growth factor (NGF). Exposure to NGF resulted in little change in
erg1 mRNA expression in PC12 cells. In contrast,
upregulation of erg2 mRNA was marked and relatively rapid
(~threefold after 1 d). No induction of erg3 gene
expression was observed after NGF treatment.
Fig. 3.
erg potassium channel mRNA
expression in PC12 cells and non-neural tissues determined by RNase
protection analysis. A, erg mRNA
expression in PC12 cells in control media or after 1, 4, or 7 d of
treatment with nerve growth factor (NGF). Brain
(Br) mRNA was used as a positive control for the
erg3 experiment. B, Expression of
erg2 and erg3 mRNA in non-neural tissues.
Retinal RNA was used as the positive control.
[View Larger Version of this Image (37K GIF file)]
Neither the erg2 nor the erg3 gene was expressed
in any of the non-neural tissues that were tested (Fig. 3B),
suggesting that expression of these genes is nervous system-specific.
This is in marked contrast to the erg1 gene, which is
expressed in several non-neuronal tissues, including adrenal gland,
thymus, and lung (Wymore et al., 1997).
Kinetic properties of the erg2 and erg3
potassium channels
The kinetic properties of the erg1, erg2,
and erg3 channels were compared by expressing the channels
in Xenopus oocytes. The waveform of the current elicited in
response to a depolarizing voltage step to +20 mV was markedly
different for the erg3 channel as compared with the other
two channels (Fig. 4). The
erg1 and erg2 channels were relatively slowly
activating delayed rectifiers, whereas the erg3 current had
a predominant transient component that decayed to a sustained plateau.
The kinetic properties of the erg1 channel were very similar
to those described previously (Sanguinetti et al., 1995; Spector et
al., 1996).
Fig. 4.
Current responses of the erg1,
erg2, and erg3 channels to a depolarizing
voltage step. The holding potential was 
90 mV, and the step was to
+20 mV. Recordings were from Xenopus oocytes and were
performed with two-electrode voltage clamp. Current records were
leakage-subtracted, and the capacitance artifact at the beginning of
the voltage step was blanked.
[View Larger Version of this Image (8K GIF file)]
The kinetic basis for the different waveforms was determined by
examining the activation and inactivation kinetics of the three
channels. The erg3 channel activated at significantly faster rates than did either the erg1 or erg2 channels
over the entire voltage range tested (Fig.
5: note that the time scale for the erg3 records is fivefold faster than for the other two
channels). The erg1 channel had generally faster activation
rates than did the erg2 channel. Over the time course
studied, activation of the erg1 channel was approximated
reasonably well by a single exponential. Activation of the
erg2 channel clearly required two exponentials, with the
faster component becoming predominant at more positive potentials.
Activation of the erg3 channel was clearly sigmoidal at
negative potentials. The transient component of the erg3
current became prominent at step potentials positive to 10 mV. All
three channels display a characteristic reduction in steady-state current at positive potentials, producing a negative slope
I-V relationship. The large size of the tail currents
relative to the currents elicited by the depolarizing voltage steps is
attributable to the rapid relief of steady-state inactivation after the
step back to 70 mV (see Fig. 7B).
Fig. 5.
Activation rates of the erg1,
erg2, and erg3 channels.
A, B, Current traces showing channel
activation and deactivation in response to voltage steps to various
potentials from a holding potential of 90 mV. Tail currents were
recorded at 70 mV. Note the much faster time scale for
erg3 as compared with the erg1 and
erg2 channels. Current records were leakage-subtracted.
C, Comparison of the activation rates of the
erg1, erg2, and erg3 channels. Activation rates were measured as the inverse of the time
constant of single or double exponentials fit to the current traces. A
single exponential gave a good fit for erg1. For
erg2, two exponentials were required, and the fraction
of the fast component is shown in the inset. For
erg3, activation was clearly sigmoidal at negative
potentials. In these cases the activation time course was fit with a
single exponential after a delay, to allow for direct comparison with
the other two channels. Data are averages from seven or eight cells;
error bars are SEM.
[View Larger Version of this Image (30K GIF file)]
Fig. 7.
Steady-state kinetic properties of the
erg1, erg2, and erg3
channels. A, Peak conductance-voltage curves were
measured by stepping to the test potential from a holding potential of
90 mV, followed by a step back to the holding potential. The sizes of
the tail currents after recovery from inactivation were used as a
measure of channel activation during the test step. The step duration
was 5 sec for erg1 and erg2 and 1 sec for
erg3. Data points are the average of seven or eight
cells and were fit with the Boltzmann equation:
G/Gmax = 1/(1 + exp
((V Vh)/kh)),
where Vh = 21 ± 1.0, 3.5 ± 0.6, and 44 ± 1.4 mV and kh = 7.6 ± 0.4, 8.3 ± 0.3, and 7.2 ± 0.2 mV for
erg1, erg2, and erg3,
respectively. The open circle represents
erg1, the filled triangle represents erg2, and the filled circle represents
erg3. B, Rectification factor or
steady-state inactivation curve. This was measured by using a protocol
similar to that described previously (Sanguinetti et al., 1995).
Channels were fully activated by stepping to +40 mV for 1 sec. Then the
fully activated I-V relationship was determined by
stepping back to various test potentials. Tail currents were extrapolated back to t = 0 to correct for
deactivation where necessary. Slope conductance was determined from the
I-V plot between 140 and 120 mV, and then the
rectification factor was calculated with the following formula:
R = I/(Gslope
(Vm EK)), where R is the
rectification factor. Data points are the average of three or four
cells and were fit with the Boltzmann equation.
Vh = 101 ± 2.4, 105 ± 0.3, and 100 ± 3.0 mV and kh = 28 ± 0.7, 27 ± 0.2, and 43 ± 1.2 mV for erg1,
erg2, and erg3, respectively.
C, Calculated steady-state conductance-voltage
curve. This was calculated by multiplying the fit conductance-voltage
and rectification factor curves together for each channel. The
dashed line corresponds to Vm = 35 mV, which is the threshold for spike initiation in a typical
sympathetic neuron. D, Normalized steady-state
conductance-voltage curve. This was measured by calculating the
steady-state conductance-voltage curve and then normalizing to the
tail current at 90 mV after complete activation of the current by a
step to +40 mV. The tail current was extrapolated back to
t = 0 to correct for deactivation. This procedure
normalized for different levels of expression between different oocytes
and different channels. Although this procedure did not give absolute
values for the fractional conductance, it did allow direct comparison
of the relative heights and shapes of the G-V curve for
the three channels. Symbols have the same representations as in
A and B. Data points are averages from
six or eight cells; error bars are SEM.
[View Larger Version of this Image (31K GIF file)]
The erg1 channel and the corresponding native
IKr current are known to have very unusual
inactivation kinetics, with the inactivation rate being significantly
faster than the activation rate at most membrane potentials (Shibasaki,
1987; Smith et al., 1996; Spector et al., 1996). For this reason, we
compared the inactivation rates of the three channels, using the triple
pulse protocol described previously for erg1 (Smith et al.,
1996; Spector et al., 1996). The inactivation process could be well
described by a single exponential for all three channels. Inactivation
of the erg1 and erg2 channels had virtually
identical time courses over the entire range of potentials tested (Fig.
6). In marked contrast, the
erg3 channel inactivated significantly more slowly at
membrane potentials positive to 40 mV.
Fig. 6.
Inactivation rates of the erg1,
erg2, and erg3 channels.
A, Current traces showing channel inactivation at 0, +20, and +40 mV. Membrane potential was depolarized to +40 mV for 1000 msec to activate the channels fully. For erg1 and
erg2, a brief (15-20 msec) hyperpolarizing step to 95
mV was used to allow for recovery from inactivation before the
depolarizing voltage step shown in the recordings. For
erg3, because the rate of deactivation was significantly
faster than for the other two channels, a slightly modified protocol
was used. The hyperpolarizing step was shorter (7 msec), and the step
potential was more positive (70 mV). With the use of this protocol
minimal deactivation (<6%) occurred so that during the subsequent
depolarization step the kinetics of inactivation were not significantly
contaminated by reactivation. B, Comparison of the
inactivation rates of the erg1, erg2, and erg3 channels. Inactivation rates were measured as the
inverse of the time constant of a single exponential fit to the current traces. Data are averages from seven cells; error bars are SEM.
[View Larger Version of this Image (11K GIF file)]
It can be seen from these data that the appearance of a transient
waveform at positive potentials in the erg3 currents, but not the erg1 and erg2 currents, is attributable
to differences in the relative rates of activation and inactivation.
The ratio between the inactivation and activation rates of the
erg3 channel was close to two at positive potentials,
whereas inactivation rates were at least 10-fold faster than activation
rates for both the erg1 and erg2 channels in this
potential range. For the erg1 and erg2 channels,
the inactivation process is in quasi-steady-state relative to the much
slower activation process. The transient waveform of the
erg3 current is produced by much the same mechanism as for a
typical "A current," which has an inactivation rate that is similar
or somewhat slower than the activation rate. The primary difference
between the erg3 current and a typical A current is the
persistence of a maintained plateau current, which is attributable to
the very shallow steady-state inactivation curve of the erg3 channel.
There were other differences among the three erg channels
that are of potential relevance to the physiological function of these
channels. The peak conductance-voltage relationship was significantly
different for each of the three channels (Fig.
7A). The midpoint for
activation shifted ~20 mV among the three channels, from 44 ± 1.4 mV for erg3 to 20 ± 1.0 mV for erg1
to 3.5 ± 0.6 mV for erg2 (n = 7 or
8). The erg1 and erg2 channels had generally similar steady-state inactivation, or rectification properties, whereas
the slope of the rectification curve for the erg3 channel was significantly shallower, although the midpoint was similar to the
other channels (Fig. 7B). This difference in slope meant that the erg3 channel had less steady-state inactivation at
potentials positive to 100 mV than did the other two channels.
The physiological function of the erg channels in the
nervous system is currently obscure, although the Drosophila
erg channel clearly acts to reduce neuronal excitability (Titus et
al., 1997; Wang et al., 1997). We examined the potential contribution
of the erg channels to the steady-state current around the
threshold for spike initiation (approximately 35 mV) in two
independent ways. Initially, the fraction of channels activated at each
potential (Fig. 7A) was multiplied by the fraction of
channels inactivated at that potential (Fig. 7B) to give the
fraction of channels open at steady-state (Fig. 7C). A
second, more direct approach was to measure the conductance at the end
of a sustained voltage step; then, to normalize for differences in
expression between different oocytes and channels, the conductance at
each potential was divided by the maximum conductance to give a
normalized steady-state conductance (Fig. 7D). There was
good agreement between the two methods used for calculating the shape
and relative peaks of the steady-state conductance (compare Fig. 7,
C and D).
The steady-state conductance or "window current" was large for the
erg3 channel, whereas the erg1 and
erg2 channels passed significantly less current. Below 35
mV, the region important for control of spike initiation, the
difference was even more striking, with the steady-state
erg3 conductance close to a maximum and the erg1
and erg2 conductances at relatively low values.
Pharmacological properties of the erg2 and
erg3 potassium channels
A characteristic pharmacological property of the
IKr current, which is thought to be encoded by
the erg1 gene, is its sensitivity to methanesulfonanilides
(Sanguinetti and Jurkiewicz, 1990). The sensitivity of all three
channels to blockade by the methanesulfonanilide E4031 was determined
(Fig. 8). The KD
for blockade by E4031 was similar for all three channels (99, 116, and
193 nM for erg1, erg2, and
erg3, respectively), which is consistent with the high degree of conservation in the pore region of these channels. As has
been described previously for the blockade of the erg1
channel by other methanesulfonanilide drugs (Snyders and Chaudhary,
1996; Spector et al., 1996), E4031 acted as an open channel blocker. The on-rate for binding was apparently very slow, and repeated depolarizing pulses were required to reach quasi-steady-state for drug
binding, particularly at low drug concentrations.
Fig. 8.
Inhibition of the erg1,
erg2, and erg3 channels by the
methanesulfonanilide E4031. A, Tail currents in the
presence of increasing concentrations of E4031. The procedure used to
measure the degree of blockade at each drug concentration was similar
to that described previously (Snyders and Chaudhary, 1996). Because the
drug is an open channel blocker with a very slow on-rate of binding, it was necessary to depolarize the cell repetitively to reach equilibrium binding. A 20 sec step to +20 mV was applied at 0.033 Hz until no
further reduction in current was seen for that particular drug concentration. At that point a test step to +10 mV for 1 sec was applied, and the tail current at 60 mV was measured.
B, Hill plots of E4031 inhibition of the
erg1, erg2, and erg3
channels showing the KD for channel
blockade. Data points were fit with the Hill equation: percentage of
blockade = 1/(1 + (KD/[E4031])), where
KD = 99 ± 10, 116 ± 11, and
193 ± 18 nM for erg1,
erg2, and erg3, respectively. Data points
are averages from three or four cells; error bars are SEM.
[View Larger Version of this Image (21K GIF file)]
DISCUSSION
In this paper we describe the identification, mRNA distribution,
and biophysical properties of two new members of the erg potassium channel gene family: erg2 and erg3.
Both genes are expressed exclusively in neural tissue, in marked
contrast to the previously described erg1 gene, which is
expressed in a wide range of tissues in addition to the nervous system
(Wymore et al., 1997). The erg3 gene is expressed abundantly
in brain and sympathetic ganglia. Distribution of erg2 gene
expression is restricted considerably more and is located primarily in
a subset of sympathetic ganglia known as the prevertebral ganglia.
The physiological role of the erg channels in the mammalian
nervous system is currently uncertain. In Drosophila it has
been shown that mutations in the erg gene produce a
hyperexcitable phenotype (Titus et al., 1997; Wang et al., 1997),
suggesting that the erg channel inhibits neuronal
excitability. The biophysical properties of the Drosophila
erg channel have yet to be characterized, however, and the native
current produced by the erg channels has not been described.
A current with pharmacological and kinetic properties that are very
similar to those of erg channels has been described in a rat
dorsal root ganglia cell line (Farvelli et al., 1996). Pharmacological
blockade of this current results in a significant decrease in spike
frequency adaptation and an increase in excitability in response to
maintained depolarizing stimuli (Chiesa et al., 1997), suggesting that
erg channels can contribute to the control of neuronal
excitability in some mammalian cells. The observation that all three
erg channels are very sensitive to blockade by
methanesulfonanilides such as E4031 should make it possible to
determine the function of the erg channels in the nervous
system, although the relatively slow onset and voltage dependence of
binding means that some caution will be required in interpreting
negative results.
Several differences in the functional properties of the three
erg channels were found that could be of physiological
importance. Most obvious was the difference in the waveform of the
currents produced in response to voltage steps to positive membrane
potentials. The current produced by the erg3 channel had a
large transient component at positive potentials, whereas the other two
channels produced slowly activating currents that resembled classical
delayed rectifiers. The kinetic basis for the switch between the two
waveforms was somewhat paradoxical. For the erg1 and
erg2 channels, the delayed rectifier waveform was produced
by an increase in the inactivation rate relative to the
inactivation rate of the erg3 channel. This result is
attributable to the very unusual kinetic properties of the
erg1 and erg2 channels, which have significantly faster inactivation than activation rates (typically at least 10-fold
faster). The activation rate was significantly slower for the
erg1 and erg2 channels than for the
erg3 channel, and this difference also contributed to the
production of different waveforms.
A more subtle difference, which is of potential importance for the
physiology of the cells that express erg channels, was the
difference in the position and peak of the steady-state
conductance-voltage curve of the three channels. The erg3
channel activated at relatively negative membrane potentials and
produced a large window current, which peaked around the threshold for
spike initiation in a typical sympathetic neuron (Wang and McKinnon,
1995). The erg3 channels could, therefore, produce a
significant inhibitory influence on subthreshold electrical
excitability. The conductance-voltage curve for the erg1
and erg2 channels was shifted to the right, and the peak
steady-state conductance was significantly lower relative to the
erg3 channel. This makes it less likely that these channels
could influence the threshold firing properties of neurons, at least as
homomultimers. In cardiac myocytes the action potential duration is
relatively long, giving the erg1 channels sufficient time to
activate fully and contribute to action potential repolarization. The
relatively brief duration of the typical neuronal action potential means that there would be relatively little activation of the erg1 or erg2 channels during a single action
potential. It is possible that the very slow deactivation kinetics of
these channels could result in cumulative activation of the channels
during a prolonged burst of action potentials. Even in this case,
however, the relatively positive activation threshold of these channels would limit their influence on neuronal excitability. It is possible, or even likely, that the functional properties of the erg1
and erg2 channels are modified by heteromultimer formation.
The broad expression of the erg3 channel in the nervous
system makes it an obvious candidate, but other, yet to be identified,
subunits also could modify the kinetic properties of the channels
significantly, as has been shown recently for the KvLQT1 channel
(Barhanin et al., 1996; Sanguinetti et al., 1996b).
The discovery that in mammals there is a small family of erg
genes may have implications for the etiology of one form of the human
genetic disease known as long QT syndrome (LQT). It has been shown that
three forms of the autosomal dominant LQT syndrome (Romano-Ward
syndrome) are produced by mutations in ion channel genes expressed in
the heart. LQT1 syndrome involves mutations in the KvLQT1 gene, which
encodes a slowly activating K+ channel that probably
underlies IKs in cardiac myocytes (Barhanin et
al., 1996; Sanguinetti et al., 1996b). LQT2 syndrome involves mutations
in the erg1 (or HERG) gene, which encodes the
IKr channel (Sanguinetti et al., 1995; Trudeau
et al., 1995), and LQT3 syndrome involves mutations in the cardiac
sodium channel gene SCN5 (or hH1) (Wang et al., 1995). Autosomal
dominant LQT syndrome characteristically displays no overt neurological
symptoms (Schwartz et al., 1994). For the KvLQT1 and SCN5 genes this
can be explained by the fact that these two genes are not expressed in
the nervous system (Gellens et al., 1992; Wang et al., 1996). For LQT2,
however, the situation is more complex, because the erg1
gene is expressed widely in the nervous system (Wymore et al., 1997).
Given this distribution pattern, the apparent lack of neurological
symptoms in LQT2 patients is somewhat surprising. Identification of the
erg2 and erg3 genes provides a potential
explanation for this paradox. The cardiac-specific nature of LQT2
syndrome may be explained by the fact that the erg1 gene is
the only member of the erg gene family expressed in heart,
and, for this reason, cardiac function may be unusually dependent on
the presence of functional erg1 channels. In contrast, in
neural tissues, two other erg genes are expressed. Although the channels encoded by these genes are not identical in function to
the erg1 channel, they are sufficiently similar to suggest that they could compensate for the reduction in functional
erg1 channels.
The observation that the erg1 gene is expressed in
sympathetic ganglia does, however, raise an interesting question
regarding the genesis of the LQT2 form of LQT syndrome. There have been two hypotheses proposed to account for the underlying etiology of LQT
syndrome (Schwartz et al., 1994; Roden et al., 1996). One hypothesis
relies on the observation that the life-threatening arrhythmias
characteristic of this disease are induced by increased sympathetic
outflow stimulated by intense emotional or physical stress. This
hypothesis has received support from the finding that the arrhythmias,
syncope, and sudden death that are characteristic of the disease can be
prevented in large part by 
-blockade and/or cardiac sympathetic
denervation. The most specific form of this hypothesis invoked a
sympathetic imbalance, positing that there was excessive left
sympathetic outflow to the heart (Schwartz et al., 1994). The second
hypothesis holds that the defect is intrinsic to the heart, affecting
the intrinsic electrophysiological function of cardiac myocytes. In the
case of LQT1 and LQT3 the intrinsic defect hypothesis is likely to be a
sufficient explanation for the syndrome, because the genes underlying
these two forms of the disease are not expressed in the nervous system.
For LQT2, however, because the erg1 gene is expressed
abundantly in sympathetic ganglia and adrenal glands in addition to
heart, the situation is more complex. Because erg channels
can contribute to the control of neuronal excitability (Chiesa et al.,
1997; Titus et al., 1997; Wang et al., 1997), disruption of
erg1 gene function conceivably could result in
hyperexcitability in sympathetic neurons, thereby affecting sympathetic
outflow to the heart, particularly during periods of physiological
stress. Alternatively, the adrenal glands might be unusually dependent
on normal erg1 channel function because, like the heart,
they express erg1, but not the other two erg
genes. Mutations in the erg1 gene could result in an
increase in circulating catecholamines, at least under some
physiological conditions. Either of these effects potentially could
contribute to the initiation of arrhythmias in LQT2 syndrome. These
possibilities are not exclusive of an intrinsic cardiac myocyte defect
in LQT2 and might act synergistically with such a defect. Although the
balance of evidence currently favors the intrinsic hypothesis for LQT2
syndrome, it will be of considerable importance to determine the
function of the erg channels in the sympathetic/adrenal
system to gain further insight into the etiology of the LQT2 form of
long QT syndrome.
FOOTNOTES
Received Aug. 18, 1997; revised Sept. 25, 1997; accepted Sept. 26, 1997.
This work was supported by National Institutes of Health Grants
NS-29755, NS-01718, HL-20558, and DK07521. We thank Dr. Michael Sanguinetti (University of Utah) for the gift of the
erg1 (HERG) cDNA clone and Drs. Paul Adams and Michael
Rosen for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Jane E. Dixon, Department of
Neurobiology and Behavior, State University of New York at Stony Brook,
Stony Brook, NY 11794-5230.
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