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The Journal of Neuroscience, September 15, 1999, 19(18):7742-7756
Two Types of K+ Channel Subunit, Erg1 and KCNQ2/3,
Contribute to the M-Like Current in a Mammalian Neuronal Cell
A. A.
Selyanko1,
J. K.
Hadley1,
I. C.
Wood2,
F. C.
Abogadie2,
P.
Delmas2,
N. J.
Buckley2,
B.
London3, and
D. A.
Brown1
1 Department of Pharmacology, 2 Wellcome
Laboratory for Molecular Pharmacology, University College London,
London, WC1E 6BT, United Kingdom, and 3 Cardiovascular
Institute, University of Pittsburgh Medical Center, Pittsburgh,
Pennsylvania 15213
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ABSTRACT |
The potassium M current was originally identified in sympathetic
ganglion cells, and analogous currents have been reported in some
central neurons and also in some neural cell lines. It has recently
been suggested that the M channel in sympathetic neurons comprises a
heteromultimer of KCNQ2 and KCNQ3 (Wang
et al., 1998 ) but it is unclear whether all other M-like currents are
generated by these channels. Here we report that the M-like current
previously described in NG108-15 mouse neuroblastoma x rat glioma
cells has two components, "fast" and "slow", that may be
differentiated kinetically and pharmacologically. We provide evidence
from PCR analysis and expression studies to indicate that these two
components are mediated by two distinct molecular species of
K+ channel: the fast component resembles that
in sympathetic ganglia and is probably carried by
KCNQ2/3 channels, whereas the slow component appears to
be carried by merg1a channels. Thus, the channels generating M-like
currents in different cells may be heterogeneous in molecular composition.
Key words:
potassium channels; neuroblastoma x glioma hybrid cells; M current; sympathetic neuron; Erg1; KCNQ
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INTRODUCTION |
The M current
(IK(M)) is a low-threshold, slowly
activating potassium current that exerts an inhibitory control over
neuronal excitability; this inhibition can be relieved by
neurotransmitters acting on G-protein-coupled receptors, leading to
enhanced excitability and reduced spike-frequency adaptation (Brown,
1988; Marrion, 1997). The current was originally described in
sympathetic neurons (Brown and Adams, 1980; Constanti and Brown, 1981),
and analogous currents have subsequently been identified in a variety
of other neuronal and non-neuronal cells. Because the precise kinetic
and pharmacological properties of the current vary somewhat in
different cell types, the name "M-like" is often applied to this
current family.
Recently, evidence has been provided to indicate that the channels that
generate the M current in rat sympathetic neurons are composed of a
heteromeric assembly of KCNQ2 and KCNQ3 subunits (Wang et al., 1998; see also Yang et al., 1998). These are two homologs
of the KCNQ1 (KvLQT1) channel, mutations of which
are responsible for one form of the cardiac "long QT" syndrome
(Yang et al., 1997). In contrast, KCNQ2 and KCNQ3
are restricted to the nervous system, and mutations in these channels
are associated with a form of infant epilepsy termed "benign familial
neonatal convulsions" (Biervert et al., 1998; Charlier et al., 1998;
Schroeder et al., 1998; Singh et al., 1998). However, it is not yet
known whether all M-like channels are composed of these two subunits (or homologs thereof), or whether members of other
K+ channel gene families might contribute
to the generation of M-like currents.
In the present experiments, we have attempted to identify the molecular
species of K+ channels that generate the
M-like current (IK(M,ng)) in NG108-15 mouse neuroblastoma x rat glioma cells. These currents have been particularly well characterized (Higashida and Brown, 1986; Brown and
Higashida, 1988a,b; Fukuda et al., 1988; Schafer et al., 1991; Robbins
et al., 1992, 1993; Selyanko et al., 1995). Like the channels in
sympathetic neurons, they are inhibited by transmitters acting on
G-protein-linked receptors coupled to phospholipase C (e.g., bradykinin
and M1 and M3 muscarinic
receptors) (Higashida and Brown, 1986; Fukuda et al., 1988), with
similar consequences for cell firing (Robbins et al., 1993). On the
other hand, the kinetics of IK(M,ng)
appear more complex than those of the ganglionic M current (Robbins et
al., 1992), and the two currents differ in their sensitivities to
9-aminotetrahydroacridine (cf. Marsh et al., 1990; Robbins et al.,
1992) and linopirdine (cf. Aiken et al., 1995; Lamas et al., 1997; Noda
et al., 1998). It has previously been suggested that
Shaker-type Kv1.2 channels, cloned from NG108-15 cells
(Yokoyama et al., 1989), may contribute to
IK(M,ng) (Morielli and Peralta, 1995).
However, the insensitivity of IK(M,ng)
to dendrotoxin (Selyanko et al., 1995) makes this unlikely. Instead, we
provide evidence to indicate that two different types of
K+ channel contribute to the M-like
current in NG108-15 cells: the mouse ether-a-go-go-related
gene (merg1a), also expressed in the brain (London et al., 1997), and
KCNQ2/KCNQ3, the proposed substrate for the ganglionic
current (Wang et al., 1998).
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MATERIALS AND METHODS |
Cell cultures. NG108-15 mouse neuroblastoma x rat
glioma hybrid cells, subclone BM8 (PM1), transfected to express pig
brain M1 muscarinic receptor (Fukuda et al.,
1988), were cultured and differentiated as described previously
(Robbins et al., 1992). Chinese hamster ovary (CHO) cells stably
transfected with cDNA encoding human M1
muscarinic receptors were maintained in culture as described in
Mullaney et al. (1993). Superior cervical ganglion neurons were
prepared as described previously (Owen et al., 1990) from 6-week-old
C57 mice and 14-d-old Sprague Dawley rats, and used after 1-2 d in
culture. Recordings from all three types of cell were made at room
temperature (20-22°C), under identical experimental conditions
(solutions, pipettes, etc.).
Culture and transfection of CHO hm1 cells. CHO hm1 cells are
CHO-K1 cells, previously transfected with the human
M1 receptor (Mullaney et al., 1993). Cells were
grown in 50 ml flasks at 37°C and 5% CO2. The
culture medium was  -MEM supplemented with 10% fetal calf serum, 1%
L-glutamine, and 1% penicillin/streptomycin. Cells were
split twice weekly when confluent, plated in 35 mm dishes, and
transfected 1-2 d after plating using "LipofectAmine Plus" (Life
Technologies, Gaithersburg, MD) according to the manufacturer's recommendations. Plasmids containing merg1a and CD8 cDNAs, both driven
by cytomegalovirus promoter, were cotransfected in a ratio of
10:1. Cells for patch clamping were identified by adding CD8-binding Dynabeads (Dynal, Great Neck, NY) the day after transfection. For
immunocytochemistry, a plasmid containing cDNA for jellyfish green
fluorescent protein (GFP) was used as a marker for transfection.
Reverse transcription PCR. RNA was extracted from cell lines
and superior cervical ganglia (SCG) using RNAzol B (Biogenesis Ltd.) and reverse-transcribed using oligo-dT and mouse murine leukemia
virus reverse transcriptase (Promega, Madison, WI). The oligonucleotides used to amplify the erg gene family were:
erg-s 5' CCCYTTCAAGGCMGTGTGGG and
erg-a 5' CTGGTHAGRCTGCTGAAGGT. Primers were designed such
that the amplified product spanned at least one intron to ensure
amplification products were not derived from contaminating genomic DNA.
The primers used for KCNQ PCR were KCNQs 5'ACCTGGARGCTBCTGGCTC and KCNQa
5'CCKCTYTTCTCAAAGTGCTTCTG. These primers were designed to
amplify both KCNQ2 and KCNQ3 sequences. Cycling
conditions were 95°C for 5 min and then 30 cycles of 95°C for 30 sec; 60°C for 1 min and 72°C for 1 min followed by a final step of
72°C for 10 min. Aliquots of the reaction mixture were visualized on
a 2% (w/v) Metaphor agarose (FMC BioProducts, Rockland, ME). PCR
products were cloned using the pGEM-T vector (Promega) and recombinant
plasmids sequenced using Taq polymerase, fluoresceinated dye
terminators and an Applied Biosystems 377 automated DNA sequencer.
Immunocytochemistry. This was performed using antibodies
raised against synthetic peptides corresponding to the last 14 amino acids of the C-terminal fragment of merg1 (merg1-CT) and the
first 17 amino acids of merg1b (merg1-NT), respectively. NG108-15
cells were seeded onto polyornithine-coated glass coverslips to allow immunocytochemistry. NG108-15 cells were washed in TBS (5.5 mM Tris, pH 7.4, and 137 mM
NaCl) and fixed in acetone for 20 min at room temperature. The fixed
cells were then treated with normal swine serum (1:10) in TBS for 30 min. Once excess serum was removed, the merg1-CT primary antibody was
applied at 1:1000 dilution for 1 hr at room temperature. Different
concentrations of the primary antibody were tested to optimize
immunochemical labeling and minimize nonspecific staining of the tissue
background. Bound antibodies were then detected using alkaline
phosphatase-conjugated secondary antibodies (1:500; Dako, Carpinteria,
CA), largely as described in Abogadie et al. (1997). Transfected
mammalian CHO cells were briefly washed with PBS and fixed for
20-30 min in PBS containing 4% paraformaldehyde. Fixed cells were
then rinsed with PBS, blocked for 10 min with BSA, and permeabilized
with 0.1% Triton X-100 for 5 min. Incubation with the anti-merg1
antibody and labeling were carried out as described for the NG108-15 cells.
Perforated-patch whole-cell recording. Cells were
bath-perfused with the solution of the following composition (in
mM): 144 NaCl, 2.5 KCl, 2 CaCl2, 0.5 MgCl2, 5 HEPES, and 10 glucose, pH 7.4, with Tris
base. Pipettes were filled with the "internal" solution containing
90 mM K acetate, 20 mM KCl, 40 HEPES, 3 MgCl2, 3 mM EGTA, and 1 mM CaCl2. The pH was adjusted to 7.4 with NaOH. Amphotericin B was used to perforate the patch (Rae et al.,
1991). The series resistance was not compensated because the error
introduced was reasonably small. Thus, with the electrodes used (2-3
M ), the series resistance was 6-8 M, and most of the currents
were <0.5 nA, so the voltage error would be <5 mV. As confirmation that the voltage error was small, no correlation was found between deactivation time constants and initial current amplitude.
Data acquisition and analysis. Data were acquired and
analyzed using pClamp software (version 6.0.3). Currents were recorded using an Axopatch 200A (or 200) patch-clamp amplifier, filtered at 1 kHz, and digitized at 1-4 kHz. In current-clamp experiments, currents
were injected, and membrane potential was recorded using an Axoclamp-2
amplifier. Activation curves were fitted by the Boltzmann equation:
I/I(50) = 1/(1 + exp(V1/2  V)/k), where I is current at
the test potential (estimated from the amplitudes of exponentials
backfitted to the beginning of the test step), I(50) is
current at +50 mV, V1/2 is the
membrane potential, V, at which I is equal to
1/2 I(50). Inhibition of the current was measured
from the change in the amplitude of the deactivation tail recorded at
50 mV. Each tail was fitted by one or more exponentials, and the tail
amplitude was taken as the sum of the amplitudes of all components
contributing to it after backfitting them to the beginning of the
hyperpolarizing pulse. In cells that had the erg-type
component, backfitting was necessary to exclude not only the
(relatively fast) capacity transient, but also the brief rising phase
of the tail caused by the deinactivation of erg-type channels. To include the fast component (when present) in the fit, we
had to position the fitting cursor earlier in the trace than was
appropriate for the pure erg current. This tends to skew the  values
obtained for the erg components to larger values. This may
explain the difference in slow values between the total and
WAY 123,398-sensitive currents (Table
1). Inhibition curves were fitted by the
Hill equation: Y = Ymax *
xnH/(xnH + IC50 nH),
where Ymax is the maximum inhibition,
x is the blocker concentration, nH is
the slope (Hill coefficient), and IC50 is the
concentration corresponding to the half-maximal inhibition. Individual
currents were measured and fitted using the Clampfit software, whereas
the program "Origin" (version 5.0, Microcal Software) was used for
fitting activation and inhibition curves and for creating the
figures.
Drugs and chemicals. Linopirdine (DuP 996) was obtained from
Research Biochemicals (Natick, MA). WAY 123,398 and azimilide were
kindly provided by Wyeth-Ayerst Research (Princeton, NJ) and Dr. A. Busch (DG Cardiovascular, Frankfurt, Germany), respectively. All other
drugs and chemicals were obtained from Sigma or BDH Chemicals (Poole, UK).
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RESULTS |
Fast and slow M-like (IK(M,ng)) currents
in NG108-15 mouse neuroblastoma x rat glioma cells
We recorded M-like currents
(IK(M,ng)) from 101 chemically
differentiated NG108-15 cells using perforated-patch electrodes. Cells
were carefully selected for their "neuron-like" appearance, i.e.,
large size and well-developed neuropil (Robbins et al., 1992). When
studied with the conventional M-current voltage protocol, that is, by
stepped hyperpolarization after predepolarization to approximately 20
mV (see Materials and Methods), currents showed characteristic M-like
deactivation tails. However, the time course of these tail currents
varied considerably from one cell to another. Figure
1 illustrates two extreme examples of this variation. Thus, in Figure 1A, deactivation
during a 6 sec hyperpolarizing step was very slow, with an apparent
"time-constant" of ~2 sec, whereas in Figure
1B, deactivation was complete within 2 sec.
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Figure 1.
Pharmacological separation of two components of
the M-like (IK(M,ng)) current in NG108-15
mouse neuroblastoma x rat glioma cells. M-like currents recorded in two
cells (A, B) as deactivating tail currents produced by
voltage steps from 20 mV (holding potential) to 50 mV. In each
cell, the control current had two different components, fast and slow,
which could be blocked by WAY 123,398 and linopirdine, respectively.
Insets on the right show the difference
(blocker-sensitive) currents. The current in A consisted
predominantly of the slow component (blocked by WAY 123,398), whereas
that in B was predominantly fast and blocked by
linopirdine.
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Because a number of tumor cells (including neuroblastoma cells) have
been reported to express HERG-like currents with relatively slow rates of deactivation (Bianchi et al., 1998), we wondered whether these might contribute to the long deactivation tails. We
tested this pharmacologically, using the HERG
channel-blocking drug WAY 123,398 (Spinelli et al., 1993; Faravelli et
al., 1996). As shown in Figure 1A, 10 µM WAY 123,398 blocked most of the long deactivation, leaving a residual fast component which was then eliminated by the M channel-blocking drug linopirdine (Aiken et al.,
1995; Lamas et al., 1997). In contrast, the fast-deactivating current
in Figure 1B was strongly blocked by linopirdine,
leaving a slower component that was blocked in turn by WAY 123,398. Thus, comparison of the linopirdine- and WAY-sensitive currents (Fig. 1, inserts) showed that in fact each cell had two components
to the deactivation currents, fast and slow, and that the overall time
course of current deactivation was determined by their proportion. Furthermore, both components contributed to the sustained current recorded at 20 mV.
Because 10 µM WAY 123,398 produces a complete block of
erg channels without affecting other
K+ channels such as sympathetic neuron M
channels (see below), we analyzed the fast and slow components of the
deactivation tails in more detail by recording currents in the absence
and presence of WAY 123,398. Figure 2
exemplifies the results obtained in 86 of 101 cells so examined. Here,
the control current recorded in the absence of WAY 123,398 (Fig.
2A) showed three components, a fast component with a
time constant of 76 msec, and a slower, biexponential component with
time constants of 340 msec and 2.1 sec. WAY 123,398 (Fig.
2B) eliminated the slower component, leaving only the
fast component (, 75 msec), whereas the difference (WAY-sensitive) current (Fig. 2C) showed only the biexponential slow
component (, 315 msec and 2.0 sec). Thus, the time constants of
the residual current recorded after application of WAY 123,398 and of the subtracted (WAY-sensitive) current accurately reproduced
the fast and slow components of the composite initial current. In this
cell, the fast and slow components contributed 70 and 30%,
respectively of the total tail current, and both contributed to the
steady outward current at the holding potential as judged from the
effect of WAY 123,398 on the holding current. On average, in the 86 cells expressing both currents, the fast- and slow-deactivating
components contributed ~33 and 67%, respectively, to the total tail
current (Table 1). In the other 15 cells, the tail current showed only the slowly deactivating component and was fully suppressed by WAY
123,398.
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Figure 2.
Kinetic analysis of the M-like current in a
NG108-15 mouse neuroblastoma x rat glioma cell. Total
IK(M,ng) was activated by holding at 20 mV
and then deactivated by a 6 sec step (top record) to
50 mV before (A) and after
(B) addition of the erg-channel
blocker WAY 123,398 (10 µM). In control
(A) the deactivation tail was fitted
(smooth line, superimposed) by the sum of fast ( = 76 msec, 254 pA) and two slow ( = 340 msec, 37 pA and 2124 msec, 70 pA) exponential curves. WAY 123,398 abolished both slow
components without affecting the fast component (B,
= 76 msec). C, Slow component obtained by
subtracting the record shown in B from that shown in
A was fitted (smooth line, superimposed)
by the sum of two kinetic components ( = 315 and 1972 msec).
All records were obtained from the same cell. Dashed
lines denote zero current levels.
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mRNAs for merg1 and KCNQ2 and
KCNQ3 in NG108-15 cells
The clearly distinguishable effects of linopirdine and WAY 123,398 on the tail currents illustrated in Figures 1 and 2 suggested that
these might be composite currents, resulting from the deactivation of
two different species of K+ channel: one
composed of linopirdine-sensitive KCNQ2/3 subunits, or
homologs thereof (Wang et al., 1998), and the other comprising a member
(or members) of the erg family. We therefore sought evidence for the presence of transcripts of these channels by RT-PCR (see Materials and Methods). Transcripts for both erg and
KCNQ2/3 were detected (Fig.
3).
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Figure 3.
cDNA from mouse SCG, rat SCG, and chemically
differentiated NG108-15 cells, was amplified using primers to the
erg family of potassium channel genes or primers
recognizing KCNQ2 and KCNQ3 potassium
channel genes. Amplified products were obtained from all cell types but
not from a negative control containing no template, indicating that
members of these families are expressed by these cells. Sequence
analysis revealed that the amplified product obtained from NG108-15
cDNA with primers to the erg genes was predominantly, if
not exclusively, merg1. Analysis of the amplified product obtained
using the KCNQ primers in both rat SCG and NG108-15 has
shown these cells express both KCNQ2 and
KCNQ3. M-1 kb ladder DNA size standards.
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Sequence analysis of 10 independent clones from the erg PCR showed that
each clone contained erg1 DNA sequence (data not shown), suggesting
that these cells express predominantly, if not exclusively, merg1 transcript (London et al., 1997). The
KCNQ2/3 transcript contained mRNA for both KCNQ2
and KCNQ3. It may be noted in Fig. 3 that these transcripts
were also present in dissociated neurons from mouse and rat SCG (see
also Shi et al., 1997; Wang et al., 1998).
Merg1 protein expression in chemically differentiated
NG108-15 cells
The presence of mRNA transcripts does not necessarily correlate
with translated protein products. We therefore tested for the
expression of merg1a protein in NG108 cells by immunocytochemistry using a specific antibody raised against the C-terminal fragment of
merg1 (merg1-CT; see Materials and Methods). As a control for specificity, the merg1-CT primary antibody was preincubated
overnight with a 10-fold molar excess of the immunogenic peptide.
Figure 4, A and B,
compare merg1 immunolabeling in chemically differentiated and
nondifferentiated NG108-15 cells, respectively. Strong labeling was
observed in both cell bodies and neuropil of most large chemically differentiated NG108-15 cells of the type we normally selected for
electrophysiological recording, whereas smaller cells with less
well-developed processes showed weak or no immunoreactivity. No
staining was observed in cells treated with preabsorbed antibody. Nondifferentiated cells, which normally expressed very small
IK(M,ng), showed moderate or no
staining. Interestingly, no staining was observed in dissociated mouse
SCG neurons (Fig. 4C), or over the underlying glial cell
layer. This latter (negative) finding accords with the absence of any
effect of WAY 123,398 on membrane currents recorded from these neurons
(see below). Also, no immunostaining of differentiated NG108-15 cells
was detected after exposure to an antibody raised against an N-terminal
sequence unique to the short form of merg1 (merg1b), the expression of
which is restricted to cardiac cells (London et al., 1997). This
suggests that the protein tagged by the C-terminal antibody is the
product of the long-form transcript merg1a (London et al., 1997; see
also below).
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Figure 4.
Immunocytochemical detection of merg1 protein in
differentiated NG108 cells. Immunostaining for the C terminus of merg1
in chemically differentiated (A) and
undifferentiated (B) NG108 cells, and in
dissociated mouse SCG cells (C). Note that in
A, a large NG108-15 cell (arrow) showed
labeling of strong intensity, whereas adjacent smaller and bipolar
cells were not stained. Micrographs were obtained using bright-field
optics. Scale bars, 20 µm.
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Strong immunoreactivity for merg1-CT antibody was also
detected in CHO cells transfected with merg1a cDNA. No staining was observed in untransfected cells, in cells transfected only with the GFP
plasmid, or in cells treated with preabsorbed antibody (data not shown).
Slow IK(M,ng) is mimicked by merg1a
current (Imerg1a) expressed in CHO
cells
The above results suggested that the slow component of the M-like
current in NG108-15 cells might well be carried by merg1a channels. We
tested this further by expressing merg1a cDNA in mammalian CHO cells.
Figure 5 illustrates the resultant
membrane currents. Imerg1a was
activated by membrane depolarization to equal or positive to
40 mV, but showed substantial inactivation during the (long)
depolarizing command at potentials positive to 0 mV (Fig.
5Aa). As a result, the "steady-state" current-voltage curve was "bell-shaped" (Fig. 5Ab). The time course of
this composite activation (accompanied by inactivation) could be
described by two exponentials, accelerating strongly with
depolarization (Fig. 5Ac). When the cell was hyperpolarized
after a depolarizing prepulse to +50 mV, there was a large transient
enhancement of the current, caused by removal of channel inactivation,
followed by a slower deactivation (Fig. 5Ba,b). Two
deactivation components were detected that were strongly shortened by
membrane hyperpolarization (Fig. 5Bc). The mean activation
curve deduced from tail currents followed a Boltzmann equation with
V1/2 = 5.9 ± 0.6 mV and
k = 12.2 ± 0.5 mV (Fig. 5C). However,
when individual curves were fitted, they showed a great variation in
V1/2 (range, between 27 and 13 mV;
n = 9) and small variation in k (range,
between 6.9 and 10). These results accord well with previous
observations on merg1a currents in oocytes (London et al., 1997).
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Figure 5.
Characteristics of the merg1a current
(Imerg1a) expressed in CHO cells.
Imerg1a was activated by long (8 sec)
depolarizing voltage steps from the holding level of 80 mV
(Aa) and deactivated by hyperpolarizing steps after its
full activation by a prepulse to +50 mV (Ba).
Leak-subtracted steady-state I-V relationships obtained
at the end of the depolarizing and hyperpolarizing pulses,
respectively, are shown in Ab and Bb
(open circles), and an "instantaneous"
I-V relationship obtained at the beginning of the
hyperpolarizing pulses for current deactivation is shown in
Bb (filled circles). Activation
(Ac) and deactivation (Bc) time constants
were plotted semilogarithmically, and -V
relationships were fitted by straight lines with at 0 mV and
the slope equal to 1218 ± 1 msec and 0.013 ± 0.001 mV 1 in Ac and 403 ± 1 msec
and 0.012 ± 0.0006 mV1 (filled
circles) and 3437 ± 1 msec and 0.014 ± 0.002 mV1 (open circles) in
Bc. C, Activation curve fitted by the
Boltzmann equation at V1/2 = 5.9 ± 0.6 mV and k = 12.2 ± 0.5 mV. Records in
Aa and Ba were from the same cell. In
Ab, Ac, Bb, Bc, and C the
mean data are shown (vertical lines indicate SEMs)
obtained from six and nine cells, respectively.
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We next compared the properties of
Imerg1a deactivation more closely with
those of the WAY-sensitive slow component of
IK(M,ng), using the "standard" M
current protocol, that is, currents were preactivated by holding at the
depolarized potential of 0 mV (20 mV in the case of
IK(M,ng), to avoid contamination by
other, primarily Ca2+-dependent,
K+ currents) and then deactivated by 6 sec
step commands to various negative potentials. As shown in Figure
6, there was a close correspondence between the two. There were three main differences. First, deactivation of Imerg1a was preceded by a larger
transient reactivation: this presumably reflected the greater
steady-state inactivation of Imerg1a
at 0 mV than that of slow IK(M,ng) at
20 mV. Second, the threshold for activation of
Imerg1a was ~10 mV more positive than that for slow IK(M,ng) (Fig.
6Ab,Bb): the reason for this is not known
but may simply relate to different cell types. Third, whereas both
showed a biexponential deactivation, the time constants for the two
components of Imerg1a deactivation
measured at 50 mV were ~40% of those for deactivation of the slow
IK(M,ng) measured at the same
potential (Table 1). The time constants showed a comparable voltage
dependence (Fig. 6,compare Ac, Bc), so this difference may be explained by the different activation thresholds and/or the different size of the voltage step (in four CHO cells using
a prepulse protocol, the first and the second slow deactivation values for Imerg1a were slower after a
prepulse to 20 mV compared with a prepulse to 0 mV, by 41 and 57%,
respectively).
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Figure 6.
Comparison of the slow M-like
(IK(M,ng)) current in NG108-15
mouse neuroblastoma x rat glioma cells (A) and
mouse-erg1a current (Imerg1a)
expressed in CHO cells (B). The slowly
deactivating component of IK(M,ng)
(Aa) was recorded in response to long hyperpolarizing
steps (holding level, 20 mV; step duration, 6 sec; interval, 60 sec;
increment, 10 mV). These slow IK(M,ng)
currents were obtained by subtracting the currents recorded in the
presence of 10 µM WAY 123,398 (box, right)
from those in the absence of the blocker (box, left).
For comparison (Ba), Imerg1a
is shown, obtained with a similar voltage protocol (note the holding
potential of 0 mV in Ba). Deactivation tails were fitted
by double-exponential curves (smooth lines,
superimposed). Leak-subtracted steady-state (filled
circles) and instantaneous (open circles)
I-V relationships for
IK(M,ng) (Ab) and
Imerg1a (Ba) were obtained by
measuring the current at the beginning and end of the voltage pulse.
Fast (filled circles) and slow (open
circles) time constants for deactivation of
IK(M,ng) (Ac) and
Imerg1a (Bc) were plotted
semilogarithmically against membrane potential, and
-V relationships were fitted by straight lines with
at 0 mV and the slope equal to 2134 ± 1 msec and 0.015 ± 0.0009 mV1 (filled
circles) and 15737 ± 1 msec and 0.016 ± 0.002 mV1 (open circles) in
Ac and 680 ± 1 msec and 0.011 ± 0.0008 mV1 (filled circles) and
5432 ± 1 msec and 0.012 ± 0.0007 mV1
(open circles) in Bc. In Ab,
Ac, Bb, and Bc the mean data are
shown (vertical lines indicate SEMs) obtained from 26 and 29 cells, respectively.
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For comparison, we also examined the properties of currents generated
by the short "cardiac" isoform merg1b expressed in CHO cells
(Imerg1b; n = 3; data
not shown). Whereas the voltage dependence of
Imerg1b activation and deactivation
were very similar to that of the slow
IK(M,ng) and of
Imerg1a, both activation and
deactivation of Imerg1b were several
times faster (Table 1), as previously reported in oocytes by London et
al. (1997) (see also Lees-Miller et al., 1997). Hence, and in
accordance with the lack of antibody staining mentioned above and the
absence of merg1b mRNA expression in the nervous system (London et al.,
1997), it is unlikely that merg1b channels contribute to slow
IK(M,ng).
Slow IK(M,ng) and
Imerg1a show similar pharmacology
We next compared the sensitivity of the slowly deactivating
component of the NG108-15 current
IK(M,ng) with that of CHO-expressed merg1a currents to some blocking drugs. As shown in Figure
7, both tail currents were blocked by the
anti-arrhythmic drug WAY 123,398 with equal facility
(IC50 values, 0.4 and 0.3 µM, respectively; Table
2). They were also equally sensitive to
another anti-arrhythmic drug, azimilide (IC50
values, 6.4 and 6.5 µM, respectively; Table 2;
Busch et al., 1998). Imerg1a was also
inhibited by 9-aminotetrahydroacridine (THA; IC50
value, 36 µM; Table 2), a compound that had
previously proved unexpectedly potent in inhibiting the M-like current
in NG108-15 cells (Robbins et al., 1992). In contrast, neither current was inhibited by the ganglionic M channel- and KCNQ2/3
channel-blocking agent linopirdine at concentrations up to 30 µM [Noda et al. (1998) reported an
IC50 of 36 µM against the
NG108-15 current, but this was measured from the depression of the
composite current, and the Hill slope was rather shallow, so probably
reflected its primary action on the fast current component, see
below]. Also, both currents were very insensitive to
tetraethylammonium (TEA), with IC50 values of 17 and 24 mM (Table 2). Thus,
Imerg1a provides a good match for the
slowly deactivating component of
IK(M,ng), both kinetically and
pharmacologically.
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Figure 7.
The slow component of the NG108-15 M-like current
and the merg1a current are equally sensitive to WAY 123,398. Records in
A and B show the slow component of
IK(M,ng) and
Imerg1a deactivation tail currents recorded
on stepping from 0 to 50 mV in the presence of increasing
concentrations of WAY 123,398 (0, 0.3, 1, and 10 µM).
Plots in C show mean percent inhibition of the tail
currents (open circles,
IK(M,ng); n = 4;
filled circles,
Imerg1a; n = 6).
See Table 2 for fitted parameters.
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Fast IK(M,ng) is mimicked by the M
current (IK(M)) in mouse sympathetic
neurons
As noted above (Fig. 1), the fast component of
IK(M,ng) was readily suppressed by 10 µM linopirdine. Since linopirdine blocks M
currents in sympathetic ganglia (Lamas et al., 1997; Wang et al.,
1998), this suggested that fast
IK(M,ng) might correspond to the
"true" (ganglionic-type) M current. We assessed this by comparing
fast WAY-insensitive IK(M,ng) with the
M current recorded from dissociated mouse SCG neurons. (We used
mouse neurons because (1) the parent neuroblastoma to the
NG108-15 hybrid cell line is derived from mouse neural crest; and (2)
sequence analysis of the PCR products obtained with the KCNQ
primers showed that NG108-15 cells expressed both the mouse KCNQ2 and
the mouse KCNQ3. Because we have successfully used these primers to
amplify the rat KCNQ2 and KCNQ3 genes, we conclude that NG108-15 cells
express predominantly, if not exclusively, the mouse KCNQ genes.)
Figure 8 shows families of fast
IK(M,ng) (Aa) and
IK(M) (Ba) activated by
membrane depolarization to 20 mV and deactivated by 1 sec hyperpolarizations at 30 to 100 mV. The slow component of
IK(M,ng) was eliminated using 10 µM WAY 123,398, as shown in the box in Figure
8. Fast IK(M,ng) and
IK(M) had similar voltage dependences (Fig. 8A,B) to each other, and also to slow
IK(M,ng) and
Imerg1a (compare Fig. 6). However,
unlike slow IK(M,ng), deactivation tails of fast IK(M,ng) and mouse
IK(M) were fitted with single exponential curves: these had time constants similar to each other but
much shorter than those in the slow
IK(M,ng) and
Imerg1a (Table 1).
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Figure 8.
Characteristics of the fast M-like
(IK(M,ng)) current in a NG108-15
mouse neuroblastoma x rat glioma cell (A) and the
M current (IK(M)) in a mouse SCG
neuron (B). The fast-deactivating component of
IK(M,ng) (Aa) was recorded in
the presence of 10 µM WAY 123,398 at different membrane
potentials (holding level, 20 mV; step duration, 1 sec; interval, 10 sec; increment, 10 mV). Box, Inhibition of the slow,
WAY-sensitive component in this cell: currents before and after
application of 10 µM WAY 123,398, left,
and the difference current, right. For comparison
(Ba), IK(M) is shown,
obtained with the same voltage protocol. Deactivation tails were fitted
by single exponential curves (smooth lines,
superimposed). Leak-subtracted steady-state (filled
circles) and instantaneous (open circles)
I-V relationships for
IK(M,ng) (Ab) and
IK(M) (Ba) were obtained by
measuring the current at the beginning and end of the voltage pulse.
When the time constants for deactivation of
IK(M,ng) (Ac) and
IK(M) (Bc) were plotted
semilogarithmically against membrane potential, -V
relationships were fitted by straight lines with at 0 mV and the
slope equal to 271 ± 1 msec and 0.009 ± 0.0004 mV1 in Ac and 399 ± 1 msec
and 0.013 ± 0.0009 mV1 in Bc.
In Ab, Ac, Bb, and Bc the mean data are
shown (vertical lines indicate SEM) obtained from 30 and
15 cells, respectively.
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Pharmacological comparison of fast
IK(M,ng) and mouse
IK(M)
Like fast IK(M,ng), mouse
IK(M) was unaffected by 10 µM WAY 123,398. IK(M) recorded from 5 rat SCG neurons
was also insensitive to this compound. Furthermore, both fast
IK(M,ng) and mouse
IK(M) were 1.5-2 orders of magnitude
less sensitive to THA than were slow
IK(M,ng) or
Imerg1a (IC50
values, 1.5 and 1.3 mM; Table 2). This accords
with the relative insensitivity of rat SCG
IK(M) to THA reported previously
(Marsh et al., 1990). Figure 9 shows the
responses of fast IK(M,ng) and mouse
IK(M) to linopirdine and TEA. Whereas
both currents were considerably more sensitive than slow
IK(M,ng) or
Imerg1a to linopirdine, the
neuroblastoma-glioma current was clearly more sensitive than the mouse
SCG current (IC50 values, 1.2 and 3.5 µM, respectively; Table 2). Likewise, fast
IK(M,ng) was more readily blocked than
the mouse IK(M) by TEA (see
Discussion). Nevertheless, although not completely identical pharmacologically, fast IK(M,ng) and
mouse SCG IK(M) are clearly similar
and together show an obvious difference from slow
IK(M,ng) and
Imerg1a.
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Figure 9.
Differential sensitivities of fast
IK(M,ng) and
IK(M) to linopirdine and TEA. The fast
(WAY-insensitive) component of IK(M,ng) and
IK(M) was recorded in response to 1 sec
steps from the holding level of 20 to 50 mV, in the absence and
presence of different concentrations of linopirdine (Aa,
Ab) and TEA (Ba, Bb).
C, Concentration dependences of inhibition of the two
currents by linopirdine (a) and TEA
(b). Smooth lines are the fits by
the Hill equation. For the parameters of the fit see Table 2.
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Both fast and slow IK(M,ng), and
Imerg1a, are inhibited through
activation of M1 muscarinic receptors
Activation of M1 muscarinic receptors
inhibits both the total (composite) M-like current
(IK(M,ng)) in NG108-15 cells (Fukuda et al., 1988; Robbins et al., 1991, 1993), and the M current
IK(M) in rat (Marrion et al., 1989;
Bernheim et al., 1992) and mouse (Hamilton et al., 1997) sympathetic
neurons. We therefore examined the effects of a muscarinic stimulant,
oxotremorine-M (Oxo-M; 10 µM), on each
component of IK(M,ng), as well as
Imerg1a, in M1
muscarinic receptor-transformed NG108-15 and CHO cells. Figure 10 shows that Oxo-M inhibited the slow
IK(M,ng) (A) and
Imerg1a (B). Such
inhibitions were observed in seven of eight NG108-15 cells (mean
inhibition, 42.3 ± 13.8%) and six of six
merg1a-expressing CHO cells (mean inhibition, 50.7 ± 10.8%). Inhibition of both slow
IK(M,ng) and
Imerg1a was accompanied by a
significant acceleration of their deactivation kinetics (Fig.
11): on average, the two components in
slow IK(M,ng) and
Imerg1a were shortened by 25.4 ± 10.0% and 34.6 ± 9.0% (n = 6) and 36.5 ± 4.8% and 27.8 ± 10.1% (n = 4), respectively.
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Figure 10.
Muscarinic inhibition of slow
IK(M,ng) in a NG108-15 mouse neuroblastoma
x rat glioma cell (A) and
Imerg1a expressed in a CHO cell
(B). Currents (Aa, Ba) were
produced by holding at 20 mV (A) or 0 mV
(B) and giving repeated steps (at 0.02 Hz in
A and 0.025 Hz in B) to 50 mV for 6 sec. Both steady-state currents at the holding potentials and
deactivation currents at the test potential were reduced by
bath-application of oxotremorine-M (Oxo-M; 10 µM). In an NG108-15 cell, inhibition of
IK(M,ng) was preceded by a transient
activation of a Ca2+-activated K+
current (Aa). Families of currents in b
and c were obtained in response to incremental (10 mV)
hyperpolarizing voltage steps before (b) and
during (c) action of Oxo-M. The
insert in B shows superimposed current
produced by voltage steps from 0 to 50 mV before and during the
action of Oxo-M.
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Figure 11.
Muscarinic inhibition of slow
IK(M,ng) in a NG108-15 mouse neuroblastoma
x rat glioma cell (A) and
Imerg1a expressed in a CHO cell
(B) is accompanied by acceleration of their
deactivation kinetics. Superimposed are deactivation tails obtained by
stepping to 50 mV from 20 mV (A) or 0 mV
(B) in control (Con) and in the
presence of 10 µM oxotremorine-M (Oxo).
Smooth lines are double-exponential fits with time
constants (indicated by arrows) equal to 276 and 1271 msec (Con) and 176 and 708 msec (Oxo) in
A and 92 and 398 msec (Con) and 71 and
290 msec (Oxo) in B.
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Figure 12 shows examples of inhibitions
of fast IK(M,ng) and mouse
IK(M) by Oxo-M. Such inhibitions were
observed in four of four NG108-15 cells (mean inhibition, 72.6 ± 13.8%; n = 4). As expected (Hamilton et al., 1997),
similar inhibition was consistently observed in sympathetic
neurons.
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Figure 12.
Muscarinic inhibition of fast
IK(M,ng) in an NG108-15 mouse neuroblastoma
x rat glioma cell (A) and
IK(M) in a mouse sympathetic neuron
(B). Fast-deactivating
IK(M,ng) currents (A)
are shown during 1 sec of hyperpolarization from 20 to 50
mV, before and during the action of oxotremorine-M
(Oxo-M; 10 µM). Both currents were
obtained in the presence of 10 µM WAY 123,398. (The
effect of WAY 123,398 on the total IK(M,ng)
recorded in this cell with a longer, 6 sec pulse, is shown in the
box.) Currents in a mouse sympathetic neuron were
obtained before and after addition of WAY 123,398 and WAY 123,398 + Oxo-M.
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Fast and slow IK(M,ng) control firing in
NG108-15 cells
The function of the M current is to act as a brake on repetitive
firing. Thus, inhibition of the M current in sympathetic neurons,
either by muscarinic agonists (Brown and Selyanko, 1985) or by an M
channel-blocking agent (Wang et al., 1998), is associated with
increased repetitive firing during depolarizing current injections. A
similar effect has also been reported in
M1-transformed NG108-15 cells after application
of a muscarinic agonist (Robbins et al., 1993). However, in the latter
case, it is not clear whether this results from inhibition of the fast
or slow IK(M,ng), or both. We tested
this by injecting long (7 sec) depolarizing currents into NG108-15
cells and then observing the effects of selectively inhibiting fast and
slow IK(M,ng) with linopirdine and WAY
123,398, respectively. The depolarizing currents produced a short burst of repetitive firing in 20 of 22 NG108-15 cells tested and single action potentials in the remaining two cells. Figure
13 shows that 30 µM linopirdine (which blocked the fast current
completely and inhibited the slow
IK(M,ng) by only 33%) produced a
strong reduction in spike adaptation, whereas 10 µM WAY 123,398 had a much weaker effect.
Neither linopirdine nor WAY 123,398 had any effect on spike
repolarization.
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Figure 13.
Effects of inhibiting fast and slow
IK(M,ng) on firing in NG108-15 cells.
Records show action potential trains in two NG108-15 cells (A,
B) produced by long (7 sec) depolarizing pulses (top
records) from the holding potential of 90 mV in the absence
of drugs (Aa, Ba), in the presence of 30 µM linopirdine (Ab), 10 µM
WAY 123,398 (Bb), or in the presence of both linopirdine
and WAY 123,398 (Ac, Bc).
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DISCUSSION |
The main point emerging from this work is that the M-like current
in NG108-15 cells (IK(M,ng)) is a
composite current generated by at least two channel types: a
fast-deactivating set of channels similar (but not quite identical) to
those carrying the M current in mouse sympathetic neurons and
tentatively identified as KCNQ2/KCNQ3 (Wang et
al., 1998), and a slower-deactivating current probably formed from
merg1a (London et al., 1997). KCNQ2 and KCNQ3 are analogs of KCNQ1, which coassembles with KCNE
(minK) subunits to give the cardiac current
IKs (the slow component of the cardiac "delayed rectifier"), and mutation of which causes one form of the
cardiac long QT syndrome (Yang et al., 1997). KCNQ2
and KCNQ3 have so far been detected only in brain and
ganglia, and are implicated in a form of juvenile epilepsy (Biervert et
al., 1998; Charlier et al., 1998; Schroeder et al., 1998; Singh et al.,
1998; Yang et al., 1998). Merg1a is one isoform of the mouse homolog of
erg, originally cloned from a rat brain hippocampal cDNA
library (Warmke and Ganetzky, 1994); mRNA for erg is
found mainly in heart and brain (London et al., 1997; Wymore et al.,
1997). Mutations of the human homolog HERG give rise to a
cardiac long QT syndrome (Curran et al., 1995; Sanguinetti et al.,
1996), whereas mutations in Drosophila erg are responsible
for the seizure phenotype associated with hyperactivity in the flight
motor pathway (Titus et al., 1997; Wang et al., 1997). Thus, both these
channel types have been implicated in the control of excitability
in vivo. This is the first example of these two channels
forming overlapping and functionally similar components of membrane current.
Our conclusion that two different channels are involved is based on:
(1) the presence of mRNA transcripts and protein; (2) biophysical
properties (voltage threshold and deactivation parameters); (3)
sensitivity to potassium channel blockers; and (4) modulation of the
channel by an agonist. Thus, we demonstrate the presence of mRNA and
protein for merg1, and mRNA for KCNQ2 and
KCNQ3, in NG108-15 cells. We show that the kinetics of
merg1a heterologously expressed in mammalian cells correspond closely
to those of the slow IK(M,ng). We also
show that the two are equally sensitive to the
merg-selective blocking agents WAY 123,398 and azimilide, and insensitive to the ganglionic M current and KCNQ2/3
channel-blocking agent linopirdine. On the other hand, we find that the
fast IK(M,ng) kinetically matches the
mouse SCG IK(M), that these two are
pharmacologically similar (although not quite identical), and that they
can be distinguished from both slow
IK(M,ng) and
Imerg1a by their greater sensitivity to linopirdine and insensitivity to WAY 123,398. We also show that both
fast and slow components of IK(M,ng)
are inhibited by stimulating M1 muscarinic
acetylcholine receptors and that the nature of the inhibition of these
two components matches that for inhibition of mouse
IK(M) and
Imerg1a, respectively.
IK(M) in rat ganglion cells has been
ascribed to current through channels composed of heteromultimeric
assemblies of expressed KCNQ2 and KCNQ3 subunits
(Wang et al., 1998). This may also be true for
IK(M) in mouse ganglion cells and for
the fast component of IK(M,ng) in
NG108-15 cells, because we have found that both cell types show
transcripts for these subunits. However, as yet, we cannot exclude a
contribution from other, homologous, KCNQ subunits.
Furthermore, fast IK(M,ng) was
distinctly more sensitive to TEA and to linopirdine than was mouse
ganglion IK(M), suggesting that the
subunit composition of the presumed-KCNQ channels in these
two cell types might differ.
The presence of functional merg1a channels in these cells is not, in
itself, particularly surprising, because mRNA transcripts have been
identified in a number of neuroblastoma-derived cells (including
NG108-15) using probes to the human homolog HERG (Bianchi et al., 1998), and an "inwardly rectifying" current,
retrospectively similar to an erg current, was reported in
NG108-15 cells by Hu and Shi (1997) (see also Bianchi et al., 1998).
However, in previous experiments, the properties of this current were
mostly studied using solutions containing a raised
K+ concentration, so that its relation to
the M-like current was difficult to discern. It is clear from the
present experiments (using normal external
K+ concentrations) that the activation
range of Imerg1a overlaps that of the
true IK(M), but that deactivation of
Imerg1a contributes a distinctive slow
component to composite current deactivation; and furthermore, that the
proportional contributions of Imerg1a and IK(M) to the total M-like current
vary appreciably from cell to cell.
The merg1a and (presumed) KCNQ currents also overlap
functionally. Thus, Chiesa et al. (1997) have provided evidence that erg channels play a role in spike frequency adaptation in
another neuroblastoma-derived cell line, not dissimilar to the role of ganglionic M channels (Jones and Adams, 1987; Brown, 1988). In the
present experiments, it appeared that inhibition of the fast (M)
channels had more effect on the response of NG108-15 cells to a
sustained current injection than did inhibition of the slow (merg)
channels (Fig. 13). However, the relative contribution of these two
currents to spike frequency adaptation may depend on the nature of the
testing pulse protocol, because merg currents activate and deactivate
more slowly than KCNQ currents, and hence accumulate during
repetitive depolarization (Schonherr et al., 1999).
The transduction mechanism for M1-mediated
inhibition of KCNQ2/3 and merg1 is still unknown.
Inhibition of Imerg1a and slow IK(M,ng) was accompanied by
accelerated deactivation, which may indicate the involvement of protein
kinase C (PKC); in HERG currents expressed in
Xenopus oocytes, similar acceleration produced by thyrotropin-releasing hormone receptor activation was mediated by PKC (Barros et al., 1998). This would accord with an earlier proposal regarding the mechanism of inhibition of the M-like current in
NG108-15 cells by bradykinin (Higashida and Brown, 1986). Although M1 receptor activation in NG108-15 cells
produces a strong elevation in intracellular
[Ca2+] (Robbins et al., 1993), it is
unlikely that Ca2+ could be a messenger
for muscarinic inhibition of slow
IK(M,ng) or
Imerg1a because, in CHO cells, the
Ca2+ ionophore ionomycin (5 µM) produced an insignificant reduction in
Imerg1a (to 89.4 ± 9.7% of
control; n = 3). As a control for the effectiveness of
ionomycin in these cells, it caused a complete block of Kv1.2 channels
expressed in CHO cells when recorded in perforated-patch or
cell-attached configurations; direct application of 500 nM Ca2+ blocked
Kv1.2 channels when recorded in the inside-out configuration (A. A. Selyanko and J. K. Hadley, unpublished observations).
Do products of erg genes contribute to M-like currents in
other neurons? Transcripts for merg1 (London et al., 1997), and for the
rat homologs erg1 and erg3 (Shi et al., 1997; Wymore et al., 1997) are
present in mammalian brain. Furthermore, both expressed merg1a currents
and the slow, presumed-merg1a component of the M-like current in
NG108-15 cells were inhibited by stimulating M1
muscarinic receptors, so they could contribute to
muscarinic-inhibitable M-like currents previously recorded in central
neurons. True, the presence of mRNA transcripts may not betoken the
assembly of functional channels: thus, no appropriate
erg-like component of membrane current could be recorded
from mouse or rat sympathetic neurons, in spite of the presence of
mRNAs (Shi et al., 1997; see also this paper), nor could we detect
merg1 immunoreactivity. However, this may not be the case for other
mammalian neurons. For example, the M-like current recorded from
isolated rat cortical neurons has been reported to be an order of
magnitude less sensitive to linopirdine than either the ganglionic or
hippocampal cell current (Noda et al., 1998; cf. Aiken et al., 1995;
Lamas et al., 1997; Schnee and Brown, 1998). Although other
explanations are possible, our findings suggest that this might arise
from a contribution by erg channels to the cortical neuron
current. In view of the significance of M-like channels as potential
targets for cognition-enhancing drugs (Zaczek and Saydoff, 1993),
further information regarding the degree of heterogeneity in the
molecular composition of the channels underlying M-like currents in
different neurons would be helpful.
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FOOTNOTES |
Received May 18, 1999; revised June 28, 1999; accepted July 2, 1999.
B.L. was supported by a Grant-In-Aid from the American Heart
Association, and the other authors were supported by the United Kingdom
Medical Research Council and the Wellcome Trust. We thank Misbah
Malik-Hall, Brenda Browning, and Mariza Dayrell for tissue culture and
Svjetlana Miocinovic (Biology Program, CalTech, Pasadena, CA)
for participation in some experiments.
Correspondence should be addressed to Dr. A. A. Selyanko,
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
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TOP
ABSTRACT
INTRODUCTION
