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J. Biol. Chem., Vol. 275, Issue 31, 24089-24095, August 4, 2000
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From the Zentrum für Molekulare Neurobiologie Hamburg,
Hamburg University, Martinistrasse 85, D-20246 Hamburg, Germany
Received for publication, April 17, 2000, and in revised form, May 16, 2000
KCNQ2 and KCNQ3, both of which are mutated in a
type of human neonatal epilepsy, form heteromeric potassium channels
that are expressed in broad regions of the brain. The associated
current may be identical to the M-current, an important regulator of
neuronal excitability. We now show that the RNA encoding the novel
KCNQ5 channel is also expressed in brain and in sympathetic ganglia where it overlaps largely with KCNQ2 and KCNQ3. In addition, it is
expressed in skeletal muscle. KCNQ5 yields currents that activate slowly with depolarization and can form heteromeric channels with KCNQ3. Currents expressed from KCNQ5 have voltage dependences and
inhibitor sensitivities in common with M-currents. They are also
inhibited by M1 muscarinic receptor activation. A KCNQ5 splice variant
found in skeletal muscle displays altered gating kinetics. This
indicates a molecular diversity of channels yielding M-type currents
and suggests a role for KCNQ5 in the regulation of neuronal excitability.
Mutations in all four known KCNQ potassium channel
genes cause human inherited disease. Mutations in KCNQ1
(also known as KvLQT1) lead to cardiac arrhythmias in the
long QT syndrome (1) that are associated with congenital deafness in
the recessive Jervell and Lange-Nielsen syndrome (2). Mutations in
either KCNQ2 or KCNQ3 lead to benign familial
neonatal convulsions (BFNC),1
a neonatal generalized epilepsy (3-5). Finally, mutations in KCNQ4 cause autosomal dominant progressive hearing loss
(6).
The expression of KCNQ2 and KCNQ3 appears to be restricted to the
nervous system (3, 7). Both subunits are co-expressed in many areas of
the brain, including the cortex, hippocampus, and thalamus (7, 8),
suggesting the formation of heteromeric KCNQ2/3 channels. This was
supported by functional expression in Xenopus oocytes.
Currents obtained by co-expressing both subunits were more than 10-fold
larger than those obtained from homomeric channels (7, 9, 10), and
dominant negative mutations in either subunit suppressed currents from
heteromeric channels (7). Currents of KCNQ2/3 heteromers resemble
M-currents (11) in their voltage dependence, kinetics, and sensitivity
to inhibitors such as linopirdine and XE991 (10, 12). M-currents are
defined by their electrophysiological and pharmacological
characteristics. These potassium currents are already present at the
slightly depolarized potentials at the threshold of action potential
firing and are highly regulated by several second messenger pathways
(13). M-currents are inhibited by the activation of muscarinic
receptors (11, 13-15). Since regulation of M-currents provides a means to set the firing rate of neurons, it is plausible that a loss of
M-current leads to neuronal hyperexcitability and epilepsy in BFNC.
Moderate changes in the magnitude of these currents may have profound
effects. It was estimated from heterologous expression of channel
mutants found in BFNC that a 25% loss of KCNQ2/KCNQ3 current may
suffice to cause epilepsy (7).
As "M-currents" are defined by a rather broad set of biophysical
and pharmacological characteristics, KCNQ2 and KCNQ3 subunits may not
be the only molecular basis for these currents. Indeed, eag-related channels share some features with M-type
currents, but opinions differ as to whether they really qualify as
M-currents (16-19). Furthermore, the voltage and time dependence of
KCNQ4 is similar to that of KCNQ2/3 channels, and KCNQ4 is also
inhibited by blockers of M-currents such as linopirdine, albeit with
lower efficiency (6). The sensitivity to these inhibitors was increased in KCNQ3/4 heteromers, suggesting that channels containing KCNQ4 may
underlie some forms of M-currents (6). Furthermore, KCNQ4 is also
inhibited by muscarinic stimulation (20). In contrast to KCNQ2 and
KCNQ3, however, KCNQ4 plays an important role in hearing (6), and its
expression in the central nervous system is restricted to some areas of
the brainstem. This includes nuclei and tracts of the auditory pathway
(21).
Cloning and Functional Expression of KCNQ5--
A partial
cDNA encoding a novel KCNQ channel fragment was isolated by
screening a human thalamus cDNA library
(CLONTECH) using a KCNQ3 cDNA as a probe. It
was extended by 5'- and 3'-rapid amplification of cDNA ends
techniques using a Marathon kit (CLONTECH) of human adult brain cDNA. The KCNQ5 gene was localized on human
chromosome spreads using fluorescent in situ hybridization
by Genome Systems. The KCNQ5 cDNA (all splice variants of Fig.
1B) was cloned into pTPN, a derivative of the
Xenopus oocyte expression vector pTLN (22) which contains an
additional PacI site 3' to the NruI site. After
linearization with HpaI, capped cRNA was transcribed using SP6 RNA polymerase in the mMessage mMachine kit (Ambion).
Xenopus oocytes were prepared by collagenase treatment,
injected with 10 ng of cRNA, and incubated for 2-3 days at 17 °C. A
dominant negative KCNQ5 mutant (G278S) and a KCNQ3 mutant (T323Y) were constructed by recombinant PCR. In some experiments, KCNQ5 (or the
G278S mutant) was co-expressed at a 1:1 ratio with KCNQ2 or KCNQ3, or
the respective dominant negative mutants KCNQ2(G279S) and KCNQ3(G318S)
(7), or the KCNQ3(T323Y) mutant using again a total cRNA amount of 10 ng per oocyte. The human M1 receptor cDNA was obtained by PCR
amplification from human fetal brain cDNA
(CLONTECH) and was cloned into the pTPN vector. 10 and 5 ng of KCNQ5 and M1 receptor cDNAs, respectively, were
co-injected into Xenopus oocytes. Conventional two-electrode
voltage clamp measurements were performed at room temperature in ND96
saline (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, pH 7.4) using a Turbotec (npi Instruments)
amplifier and pClamp (Axon Instruments) software. For cation
selectivity measurements, NaCl was replaced by equimolar amounts of the
respective chloride salt. When using TEA-chloride, this inhibitor
replaced equivalent amounts of NaCl. For experiments examining the
effect of muscarinic receptor stimulation, 1.6 mM
Ca2+ was replaced by Mg2+ to reduce the
activation of endogenous Ca2+-activated chloride channels.
Tail current analysis was used to estimate apparent
Popen (Fig. 4). From a holding potential of Northern Blot and in Situ Hybridization--
Multiple tissue
Northern blots (CLONTECH human 7760-1, human
brain II 7755-1, and human brain III 7750-1) were hybridized overnight
at 68 °C in 7% SDS, 10% polyethylene glycol 6000, 1.5× SSPE
(SSPE, 150 mM NaCl, 10 mM
NaH2PO4, 1 mM EDTA, pH 7.4), 100 µg/ml denatured herring sperm DNA with a 32P-labeled
AflIII/EcoRI fragment of pTPN-KCNQ5 that covers
the last 1.1 kilobase pairs of the open reading frame. Blots were washed three times for 40 min in 0,1% SDS in 0.1× SSC (SSC, 150 mM NaCl, 15 mM sodium citrate, pH 7.0) at
50 °C and exposed on X-Omat film (Eastman Kodak Co.). mRNA was
isolated from the NG108-15 cell line using the Trizol reagent (Life
Technologies, Inc.) and oligo(dT) Dynabeads (Dynal), and 1.8 µg per
lane were separated on a gel containing formaldehyde. The RNA was
transferred to Hybond-N membrane and probed with a human KCNQ3 and a
rat KCNQ5 probe. For the in situ hybridization of rat brain
sections, a 1.1-kilobase pair rat KCNQ5 fragment (obtained by PCR and
covering the equivalent region of 170-1290 base pairs of human KCNQ5)
was subcloned into pBluescript, and radioactive sense and antisense
cRNAs were transcribed (Maxiscript, Ambion) using T3 and T7 RNA
polymerase, respectively, and hydrolyzed to roughly 200-300-base pair
fragments. In situ hybridization was performed as described
(23). The rat brain sections were exposed to film, and the rat superior
cervical ganglion section was dipped in a photoemulsion.
Primary Structure and Tissue Distribution of KCNQ5--
By
homology screening of a human brain cDNA library with
KCNQ3, we isolated a novel member of the KCNQ gene family
that we named KCNQ5 (Fig. 1).
It encodes a protein of 897 amino acids that has a predicted molecular
mass of ~99 kDa. It has six transmembrane domains, a P-loop,
and a carboxyl-terminal conserved cytoplasmic region (the
"A-domain") (24) (which may be involved in subunit interactions
(25)) like the other four known KCNQ proteins with which it shares
roughly 40% overall identity. Similar to other KCNQ channel cDNAs
(3, 8, 26), we detected several splice variants in the cytoplasmic,
carboxyl-terminal tail (Fig. 1B). Most of our functional
studies were performed with variant I, which we had isolated from
brain.
By using fluorescent in situ hybridization of human
metaphase cells, the KCNQ5 gene was localized to the
long arm of chromosome 6 (6q14). Northern analysis revealed that
KCNQ5 is mainly expressed in brain and skeletal
muscle (Fig. 2A). Its mRNA
is present in many regions of the human brain, including the cerebral
cortex, occipital pole, frontal and temporal lobes, putamen, and the
hippocampus (Fig. 2B). In these regions, it is co-expressed
with KCNQ2 (3) and KCNQ3 (7). Some regions that express significant
levels of KCNQ2 or KCNQ3 (e.g. cerebellum, medulla, and
thalamus) (7, 8) express only low to undetectable levels of KCNQ5.
In situ hybridization of rat brain sections largely
confirmed this distribution of KCNQ5 within the central nervous system
(Fig. 3A). In addition, it
revealed significant expression in the piriform cortex, the entorhinal
cortex, the pontine medulla, and the facial nucleus. In apparent
contrast to the human Northern analysis (Fig. 2B), there was
also a faint signal in the rat cerebellum (Fig. 3A). A weak
expression in the rat cerebellum was subsequently confirmed by Northern
analysis (data not shown).
M-currents have often been studied in sympathetic neurons (10-12, 14,
15, 27-29). This includes neurons of the rat superior cervical
ganglion, which has recently been shown (10) to express both KCNQ2 and
KCNQ3. In situ hybridization revealed that this ganglion
also expresses KCNQ5 (Fig. 3C). A system frequently used to
study neuronal M-currents is the NG108-15 mouse neuroblastoma × rat glioma cell line. These cells were recently shown to co-express KCNQ2 and KCNQ3 (18, 19). Northern analysis revealed that these cells
also express KCNQ5 (Fig. 2C). Thus, this cell type expresses
all three subunits.
Properties of KCNQ5 Channels--
We expressed the three splice
variants of KCNQ5 (Fig. 1B) in Xenopus oocytes
and investigated the associated currents by two-electrode voltage
clamping. Since there were no conspicuous differences between variants
I and II, we focused on variants I and III that we had isolated from
brain and skeletal muscle, respectively. Both forms of KCNQ5 yielded
currents that needed several seconds to activate fully upon
depolarization (Fig. 4, A and
B). This activation was faster than with KCNQ4 (6) but
slower than with KCNQ2 (3) (see also Fig. 7B) and in
particular KCNQ1 (30, 31). Interestingly, gating kinetics differed
markedly between both splice forms. The muscle isoform III showed a
faster initial activation but did not reach steady-state activation
even after 10 s. By contrast, the brain isoform I initially
activated more slowly but reached steady-state after 2-3 s (for time
constants, see legend to Fig. 4). This difference in gating kinetics is
also apparent when a typical M-current voltage clamp protocol is
employed. Although the brain variant yields currents that kinetically
resemble M-currents (Fig. 4C), the muscle variant III does
not show the typical current relaxations after hyperpolarizing voltage
steps (Fig. 4D).
Tail current analysis was used to estimate activation parameters. Both
variants begin to activate at voltages around
We studied the pharmacology of KCNQ5 using the brain variant I (Fig.
5). Tetraethylammonium (TEA) was a poor
inhibitor with an IC50 of about 70 mM (Fig.
5A), whereas barium was more effective (>60% inhibition at
1 mM and 0 mV; data not shown). Linopirdine and XE991,
which are rather specific inhibitors of M-type currents (10, 12, 34,
35), were much more potent and inhibited KCNQ5 with an IC50
in the 50-70 µM range (Fig. 5A). This is
higher than the values reported for KCNQ2/KCNQ3 heteromers
(linopirdine, 4 µM; XE991, 0.6 µM) (10).
When comparing these results to native M-currents, however, one should
bear in mind that oocyte-expressed channels may differ in their
sensitivity to inhibitors. Additionally, we identified niflumic acid as
an activator of KCNQ5 currents. At 0.5 mM, it shifted the
apparent Popen by approximately 20 mV to
negative voltages (Fig. 5B). This resembles effects of
niflumic acid on IsK channels (36), which are heteromers of KCNQ1 and KCNE1 (30, 31).
We also co-expressed KCNQ5 with several Inhibition of KCNQ5 by M1 Receptor Stimulation--
One of the
defining features of M-currents is their inhibition via muscarinic
receptors. We therefore co-injected Xenopus oocytes with
cRNAs encoding KCNQ5 and the M1 muscarinic receptor, respectively.
Muscarinic stimulation led to a transient activation of
Ca2+-activated chloride currents that are endogenous to
Xenopus oocytes. KCNQ5 currents were measured before
muscarinic stimulation and more than 2 min after stimulating M1
receptors with either 10 µM muscarine or oxotremorine
methiodide. After this time, the Ca2+-activated chloride
currents had largely disappeared due to desensitization processes.
KCNQ5 currents were inhibited typically by more than 80% after M1
receptor stimulation (Fig. 6). Thus,
KCNQ5 also shares this type of regulation with neuronal M-currents.
Interactions with Other Neuronal KCNQ Proteins--
As several
KCNQ subunits can assemble to form heteromeric channels (6, 7, 9, 10),
we investigated whether KCNQ5 can assemble with KCNQ2 or KCNQ3 with
which it is co-expressed in many regions of the brain and in
sympathetic ganglia. We co-expressed KCNQ2 with KCNQ5 using constant
total amounts of cRNA but different ratios of cRNA concentrations to
allow for different stoichiometries of channel assembly. In none
of these co-injection experiments did the magnitude of currents
significantly differ from those elicited by either homomeric KCNQ2 or
KCNQ5 channels (Fig. 7A). Furthermore, the kinetic properties of currents could not be
distinguished from a linear superposition of both currents (Fig.
7B). This is in contrast to the large current increase upon
co-expression of KCNQ2 with KCNQ3 (7, 9, 10). As a further test, we
co-expressed KCNQ5 with a dominant negative mutant of KCNQ2 (G279S) (7) and also WT KCNQ2 with the equivalent mutant of KCNQ5 (G278S). When
expressed by themselves, these mutants did not yield currents. The
currents resulting from these co-expression experiments were approximately 50% of WT KCNQ5 or KCNQ2 currents (Fig. 7A).
This current level would be expected in the absence of an interaction because 50% of WT RNA was injected to keep the total amount of cRNA
constant.
Kinetic analysis revealed that currents of oocytes co-injected with
KCNQ2 and KCNQ5(G278S) activated like KCNQ2 currents and that
KCNQ2(G279S)/KCNQ5 injected oocytes gave currents that kinetically resembled KCNQ5 currents (Fig. 7B). This suggests that
either these subunits do not interact or that the (presumably
tetrameric) channels containing one or more mutated subunits do not
yield currents. In the latter case, however, the current observed after a 1:1 co-expression of WT and mutant subunits should be reduced to less
than 10% of WT if the subunits do not show any preference in assembly.
Since this is not the case (Fig. 7A), it is concluded that
KCNQ2 and KCNQ5 do not interact strongly.
Similar tests were applied to study a possible KCNQ3-KCNQ5
interaction (Fig. 7C). KCNQ3 by itself yields currents that
are barely above background in Xenopus oocytes (7, 10). When co-expressed with KCNQ5, KCNQ3 increased currents (by a factor of >2)
when co-injected at a low KCNQ3/KCNQ5 ratio. However, it led to a
decrease in currents when present at higher ratios (Fig. 7C). The dominant negative KCNQ3 mutant G318S (7)
significantly suppressed KCNQ5 currents. These experiments suggest that
KCNQ3 and KCNQ5 form heteromers.
A still more stringent test was performed by exploiting the
differential sensitivity of heteromers to inhibition by TEA. KCNQ5 is
poorly inhibited by TEA (Fig. 5, A and C). This
is consistent (41) with the absence of a tyrosine residue at position
283 following the highly conserved GYGDK sequence in the P-loop (Fig. 1A). Indeed, KCNQ2, which has a tyrosine at that position,
is more sensitive to TEA, whereas KCNQ3, which lacks this tyrosine, is
rather insensitive (IC50 = 224 mM) (10, 42).
When KCNQ3 and KCNQ5 were co-injected, the sensitivity to TEA was
decreased (Fig. 5C), again indicating the formation of
heteromeric channels. To support this result further, we generated a
mutant of KCNQ3 in which the threonine at position 323 was replaced by
tyrosine (T323Y). This mutant is expected to be more sensitive to TEA. However, and probably related to the fact that even WT KCNQ3 expresses poorly in Xenopus oocytes (7, 10), we could not detect
currents from KCNQ3(T323Y). Work on other potassium channels indicated that the affinity of the blocker TEA increases with the number of
tyrosines at this position within the tetrameric channel (41). Thus,
one would expect that KCNQ3(T323Y)/KCNQ5 heteromers are more sensitive
to TEA than are the corresponding WT heteromers, and this is indeed the
case (Fig. 5C). Importantly, these experiments show a change
in intrinsic properties of channels by a point mutation which do not
depend on the level of surface expression. Since KCNQ3 yields only very
small currents in oocytes, and because KCNQ3(T323Y) does not give
currents by itself, this unambiguously demonstrates the formation of
KCNQ3/5 heteromeric channels.
As KCNQ5 is co-expressed with both KCNQ2 and KCNQ3 in many brain
regions, we also co-expressed all three subunits at 1:1:1 cRNA ratio in
oocytes. Currents could not be distinguished from a superposition of
KCNQ2/KCNQ3 with KCNQ3/KCNQ5 currents (data not shown).
Our work suggests that KCNQ5, in addition to KCNQ2 and KCNQ3,
contributes to M-type potassium currents in broad regions of the brain
and in sympathetic neurons. KCNQ2 and KCNQ3 co-expression yields
currents that are at least 10-fold larger than those of KCNQ2 (7, 10).
By contrast, KCNQ5 will lead to a more moderate increase in M-currents
when co-expressed with KCNQ3 but may either increase or decrease
currents when co-expressed with both KCNQ2 and KCNQ3 in the same cell.
This is because the amount of KCNQ3 available to form the more
"efficient" KCNQ2/KCNQ3 heteromers is reduced by binding to KCNQ5.
Thus, in addition to forming a novel M-type current by itself or
together with KCNQ3, KCNQ5 may also down-regulate the number of
KCNQ2/KCNQ3 heteromers. Since all three KCNQ subunits are broadly, but
differentially, expressed in brain, there will be various types of
M-type currents in different neuron populations. Furthermore, different
M-type channels may be expressed within single neurons. This will
clearly be the case for NG108-15 cells, which have been used as a model
system to study M-currents and which co-express KCNQ2, KCNQ3, and KCNQ5.
KCNQ5 is also prominently expressed in human skeletal muscle (Fig.
2A). This is also true for the rat, in which Northern
analysis revealed KCNQ5 expression in that tissue, but probably at
lower levels (data not shown). The function of KCNQ5 in skeletal muscle is presently unclear. To the best of our knowledge, there are no
reports of M-type currents in that tissue. Given the kinetic properties
of the skeletal muscle splice variant III, which does not yield
"M-type" gating kinetics in typical voltage clamp protocols (Fig.
4D), this may not be surprising. One report stated that linopirdine (215 µM) increased the twitch tension of
mouse diaphragm muscle upon direct electrical stimulation (43).
Although the mechanism is not clear, one might speculate that this is
due to the inhibition of muscular KCNQ5 channels.
Mutations in either KCNQ2 or KCNQ3 lead to BFNC,
a neonatal human epilepsy (3-5). Mutations underlying this disorder
(which include KCNQ2 gene deletions on one allele) (5) lack
dominant negative effects (3, 7). The predicted loss of KCNQ2/KCNQ3 current is small (7). The effects of mutations in KCNQ5 on neuronal excitability are more difficult to predict and will depend on
the relative expression levels of these subunits. In cells expressing
only KCNQ5, or in cells expressing KCNQ5 and KCNQ3, a loss of KCNQ5
function would entail an increase in excitability that could lead to
epilepsy. The situation is more complex in neurons expressing KCNQ2,
-3, and -5, since KCNQ2/KCNQ3 channels yield larger macroscopic
currents than do KCNQ3/KCNQ5 heteromers. Thus, depending on the
particular situation, a loss of KCNQ5 function could lead to either an
increase or a decrease in M-current magnitude. On the other hand,
certain types of KCNQ5 mutations (e.g. dominant negative ones) could reduce overall M-type currents in neurons co-expressing all three subunits independent of their relative expression levels. Thus, KCNQ5 may be a good candidate gene
for epileptic disorders, even though no epilepsy locus has been
assigned so far to human chromosome 6q14 where KCNQ5 maps.
In addition, since M-current activators may be useful for treating
epilepsy, and because drugs inhibiting M-currents may be useful for
treating the cognitive deficits in Alzheimer's disease (12, 34, 35), the identification of KCNQ5 as a mediator of M-currents opens new
perspectives for the treatment of neurological disorders.
We thank Susanne Fehr for help with the
in situ hybridization of rat brain sections, Tatjana
Kharkovets for the KCNQ3(T323Y) mutant, and Thomas Friedrich for
cloning the M1 receptor cDNA. Work in this laboratory was supported
by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
After submission of our manuscript, Lerche
et al. (44) have also reported the cloning of KCNQ5.
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF202977.
§
To whom correspondence should be addressed. Tel.:
49-40-42803-4741; Fax: 49-40-42803-4839; E-mail:
Jentsch@plexus.uke.uni- hamburg.de.
Published, JBC Papers in Press, May 17, 2000, DOI 10.1074/jbc.M003245200
The abbreviations used are:
BFNC, benign
familial neonatal convulsions;
TEA, tetraethylammonium;
WT, wild type;
PCR, polymerase chain reaction.
KCNQ5, a Novel Potassium Channel Broadly Expressed in Brain,
Mediates M-type Currents*
, and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80
mV, oocytes were clamped to values between 100 and + 40 mV for 2 s. Since variant III does not reach steady-state activation even after
more than 10 s, the parameters obtained for this splice form do
not reflect steady-state values. Tail currents were measured at 30 mV
by extrapolating to the time when the voltage was changed. Since channel gating is slow compared with the voltage jump, these currents are proportional to the number of channels that are open at the prepulse voltage.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Primary structure of the KCNQ5
protein. A, alignment of human KCNQ1 through KCNQ5. The
brain splice variant I of KCNQ5 is shown. This includes an exon
beginning at residue 373 (marked by vertical lines
below the sequence). Residues that are identical in all five
proteins are shown on a black background. The predicted
transmembrane domains S1 through S6, the pore-forming P-loop, and the
highly conserved cytoplasmic A-domain (24) are indicated by lines
above the sequence. A consensus site for
cAMP-dependent phosphorylation is indicated by an
asterisk. B, three splice variants of KCNQ5.
Variant I (also shown in A) was found by reverse
transcriptase-PCR in brain, whereas variants II and III were found in
skeletal muscle. Variant III introduces a second protein kinase A
consensus site as indicated by the asterisk and adds six
positively charged residues. C, dendrogram of KCNQ channels
using the ClustalX program.
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Fig. 2.
. Northern blot analysis of KCNQ5
expression. A, among different human tissues,
expression of KCNQ5 is highest in brain and skeletal muscle.
B, in the human brain, KCNQ5 is broadly expressed in several
different regions. This includes prominent signals in the cortex, the
occipital pole, frontal and temporal lobes, the putamen, and the
hippocampus. C, the NG108-15 neuroblastoma × glioma
cell line, which has been extensively used as a model system to study
M-currents, expresses both KCNQ3 and KCNQ5.
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Fig. 3.
In situ hybridization of adult rat
brain sections (A) and a rat superior cervical
ganglion (C). B and D are
the corresponding sense controls. Consistent with the distribution
determined by Northern analysis (Fig. 2B), KCNQ5 is highly
expressed in the cortex, hippocampus, and the caudate putamen.
CA3, CA3 field of the hippocampus; CPu, caudate
putamen (striatum); Ent, entorhinal cortex; Pn,
pontine nucleus; Fn, facial nucleus.
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Fig. 4.
Electrophysiological properties of
KCNQ5. Both the splice variant I (found in brain) and splice
variant III (found in muscle) were expressed in Xenopus
oocytes and examined by two-electrode voltage clamping. Both variants
activate slowly upon depolarization, but form I (A)
initially activates slower than the muscle form III (B).
Starting from a holding potential of 80 mV, the voltage was stepped
for 0.8 s to values between 100 and + 40 mV in steps of 10 mV,
followed by a voltage step to 30 mV (see inset,
A). Channel activation by depolarization was fitted (for
2 s steps) by a sum of three exponential functions. For a step to
+20 mV, the rate constants were t1 = 37.2 ± 2.2 ms, t2 = 246 ± 17 ms, and
t3 = 1112 ± 91 ms for splice variant I,
whereas the constants were t1 = 24.5 ± 0.8 ms, t2 = 163 ± 6 ms, and
t3 = 1690 ± 46 ms (± S.E.,
n = 16) for variant III. This difference in kinetics
also results in different curves when a typical M-current protocol is
used (C, variant I; D, variant III). Variant I
induces currents that kinetically resemble M-currents. In this
protocol, the membrane voltage is clamped for 1 s to voltages
between 30 and 90 mV in steps of 10 mV, from a holding potential
of 30 mV. This was followed by a step to 30 mV (C,
inset). E and F, apparent open probabilities
of variants I (E) and variant III (F) as a
function of voltage obtained from tail current analysis as described
under "Materials and Methods." Mean values obtained from 12 oocytes
are shown. Fitting a Boltzmann equation yielded V1/2 = 46 ± 1 mV and an apparent gating charge of z = 2.8 ± 0.1 for isoform I and V1/2 = 48 ± 1 mV and an apparent gating charge of z = 2.5 ± 0.1 for isoform III. For this fit, values at potentials more
positive than +10 mV were excluded, as these are probably affected by a
second (inactivation) gating process that leads to a decrease of
apparent Popen at more positive potentials.
G, ion selectivity of KCNQ5 currents (variant I).
Extracellular sodium was replaced by equimolar amounts of potassium.
The reversal potential is shown as a function of the potassium
concentration. This yielded a slope of 51 mV/decade potassium
concentration, indicating a highly selective potassium channel. Data
are from 10 oocytes from 2 different batches. E
G, the
error bars are smaller than the symbols.
60 mV and show a
decrease in apparent Popen at voltages more
positive than approximately +20 mV (Fig. 4, E and
F). This may be due to an inactivation process at positive
voltages as observed, e.g. with KCNQ1 (32, 33). The apparent
half-maximal activation did not show a large difference between these
isoforms (V1/2 ~ 46 ± 1 mV and
V1/2 ~ 48 ± 1 mV for isoforms I and III,
respectively). This is more negative than with KCNQ4
(V1/2 ~ 10 mV) (6), but it is in the same range
as values for KCNQ2 (V1/2 ~ 37 mV) (3) and
KCNQ2/3 heteromers (V1/2 ~ 40 mV) (10). It fits
nicely with values measured for native M-currents in cervical
sympathetic ganglion cells (V1/2 ~ 45 mV) (10).
Ion substitution experiments (Fig. 4G) revealed that KCNQ5
currents (isoform I) are highly selective for potassium. Other
experiments (not shown) yielded a Rb+ ~ K+ > Cs+ > Na+ selectivity sequence.
View larger version (11K):
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Fig. 5.
Pharmacology of KCNQ5 and KCNQ3/5
heteromers. A, inhibition of KCNQ5 homomers by
extracellular linopirdine (down triangles), XE991 (up
triangles), and TEA (squares). IC50 values
of 51 ± 5 µM, 65 ± 4 µM, and
71 ± 17 mM, respectively, were obtained from the
plotted fit curves. B, niflumic acid alters the voltage
dependence of the apparent Popen. In the
presence of 500 µM niflumic acid (open
circles), the voltage dependence is shifted about 20 mV toward
negative potentials. C, TEA sensitivity is altered in
KCNQ3/5 heteromers. Co-expression of KCNQ5 and KCNQ3 (1:1)
(diamonds) increased the IC50 value to ~200
mM, and co-expression with the KCNQ3 (T323Y) mutant
(circles) decreased the IC50 to ~30
mM. Data points in A and C are the
means ± S.E. of 4-12 individual measurements.
Popen was determined as in Fig. 4.
-subunits of the
KCNE gene family. When we expressed KCNQ5 (variant I) with
KCNE1 (minK, IsK) (37) at levels that drastically activate and slow KCNQ1 currents (30, 31), we observed a reduction in the magnitude of
these currents that also activated slightly slower (data not shown).
The kinetic effect, however, might be explained by a superposition with
a current formed by KCNE1 with xKCNQ1 which is endogenous to
Xenopus oocytes (30). Since KCNE1 could not be detected in brain and skeletal muscle by Northern analysis (38), an interaction with KCNQ5 is unlikely to be of physiological relevance. KCNE2 (Mirp1)
(39), which shows significant expression in skeletal muscle, led to a
slightly faster activation of KCNQ5 (muscle variant III). KCNE3 (39,
40), which interacts with KCNQ1 to form constitutively open channels
(40), also suppressed KCNQ5 (variant I) currents in a
dose-dependent manner (data not shown). The lack of drastic effects on channel properties, however, raises doubts on the
physiological significance of these findings.
View larger version (10K):
[in a new window]
Fig. 6.
Inhibition of KCNQ5 currents by stimulating
M1 receptors that were co-expressed in Xenopus
oocytes. A, currents before stimulating the M1
receptor; B, currents observed 3 min after applying 10 µM muscarine. The pulse protocol is shown in the
inset of B. No effect of 10 µM
muscarine or oxotremorine methiodide was found in oocytes injected with
KCNQ5 alone (data not shown). Isoform III was similarly inhibited by
stimulating M1 receptors (not shown).
View larger version (11K):
[in a new window]
Fig. 7.
Interactions between KCNQ2 and KCNQ5
(A and B) and KCNQ3 and KCNQ5
(C). A, currents at the end of a 2-s
pulse to 0 mV from a holding potential of 80 mV of oocytes injected
with different combinations of KCNQ cRNAs (always 10 ng of total amount
of RNA). The current amplitude elicited by co-injecting KCNQ2 and KCNQ5
could be explained by a linear superposition of currents. Co-injection
of KCNQ5 with the dominant negative mutant KCNQ2(G279S) (7) or of KCNQ2
with the equivalent mutant KCNQ5(G278S) leads to a roughly 50%
reduction in current amplitude, which is consistent with a lack of
interaction since only 50% of WT cRNA was injected. B,
normalized current traces of experiments used for A.
Currents elicited by the depolarizing pulse to 0 mV are shown. KCNQ2
(labeled Q2) activates faster than KCNQ5 (Q5),
and the co-expression of both yielded currents that may be explained by
a linear superposition. Co-injecting KCNQ2 with the dominant negative
(and otherwise non-functional) mutant KCNQ5(G278S) yielded currents
that were kinetically similar to KCNQ2, and currents from a
KCNQ5/KCNQ2(G279S) co-injection resembled KCNQ5 currents. C,
interactions between KCNQ3 and KCNQ5 measured as in A. KCNQ3
yields only very small currents in Xenopus oocytes (7, 10).
Currents were enlarged when KCNQ5 was co-expressed with small amounts
of KCNQ3 and were reduced when co-expressed with larger amounts. The
dominant negative mutant KCNQ3(G318S) decreased currents significantly
below 50% of WT KCNQ5 currents that would be expected from the
injected 50% of WT KCNQ5 cRNA in this experiment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
Addendum
FOOTNOTES
Present address: Institut für Humangenetik,
Universität Bonn, Wilhelmstrasse 31, D-53111 Bonn, Germany.
ABBREVIATIONS
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TOP
ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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H. Wen, T. M. Weiger, T. S. Ferguson, M. Shahidullah, S. S. Scott, and I. B. Levitan A Drosophila KCNQ Channel Essential for Early Embryonic Development J. Neurosci., November 2, 2005; 25(44): 10147 - 10156. [Abstract] [Full Text] [PDF]
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W. Shen, S. E. Hamilton, N. M. Nathanson, and D. J. Surmeier Cholinergic Suppression of KCNQ Channel Currents Enhances Excitability of Striatal Medium Spiny Neurons J. Neurosci., August 10, 2005; 25(32): 7449 - 7458. [Abstract] [Full Text] [PDF]
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M. P. G. Korsgaard, B. P. Hartz, W. D. Brown, P. K. Ahring, D. Strobaek, and N. R. Mirza Anxiolytic Effects of Maxipost (BMS-204352) and Retigabine via Activation of Neuronal Kv7 Channels J. Pharmacol. Exp. Ther., July 1, 2005; 314(1): 282 - 292. [Abstract] [Full Text] [PDF]
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A. D. Wei, A. Butler, and L. Salkoff KCNQ-like Potassium Channels in Caenorhabditis elegans: CONSERVED PROPERTIES AND MODULATION J. Biol. Chem., June 3, 2005; 280(22): 21337 - 21345. [Abstract] [Full Text] [PDF]
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A. Schenzer, T. Friedrich, M. Pusch, P. Saftig, T. J. Jentsch, J. Grotzinger, and M. Schwake Molecular Determinants of KCNQ (Kv7) K+ Channel Sensitivity to the Anticonvulsant Retigabine J. Neurosci., May 18, 2005; 25(20): 5051 - 5060. [Abstract] [Full Text] [PDF]
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A. Peretz, N. Degani, R. Nachman, Y. Uziyel, G. Gibor, D. Shabat, and B. Attali Meclofenamic Acid and Diclofenac, Novel Templates of KCNQ2/Q3 Potassium Channel Openers, Depress Cortical Neuron Activity and Exhibit Anticonvulsant Properties Mol. Pharmacol., April 1, 2005; 67(4): 1053 - 1066. [Abstract] [Full Text] [PDF]
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A. P. Berg, E. M. Talley, J. P. Manger, and D. A. Bayliss Motoneurons Express Heteromeric TWIK-Related Acid-Sensitive K+ (TASK) Channels Containing TASK-1 (KCNK3) and TASK-3 (KCNK9) Subunits J. Neurosci., July 28, 2004; 24(30): 6693 - 6702. [Abstract] [Full Text] [PDF]
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Y. Li, N. Gamper, and M. S. Shapiro Single-Channel Analysis of KCNQ K+ Channels Reveals the Mechanism of Augmentation by a Cysteine-Modifying Reagent J. Neurosci., June 2, 2004; 24(22): 5079 - 5090. [Abstract] [Full Text] [PDF]
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C. Yue and Y. Yaari KCNQ/M Channels Control Spike Afterdepolarization and Burst Generation in Hippocampal Neurons J. Neurosci., May 12, 2004; 24(19): 4614 - 4624. [Abstract] [Full Text] [PDF]
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 M C Richards, S E Heron, H E Spendlove, I E Scheffer, B Grinton, S F Berkovic, J C Mulley, and A Davy Novel mutations in the KCNQ2 gene link epilepsy to a dysfunction of the KCNQ2-calmodulin interaction J. Med. Genet., March 1, 2004; 41(3): e35 - 35. [Full Text] [PDF]
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 N. A. Singh, P. Westenskow, C. Charlier, C. Pappas, J. Leslie, J. Dillon, V. E. Anderson, M. C. Sanguinetti, and M. F. Leppert KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum Brain, December 1, 2003; 126(12): 2726 - 2737. [Abstract] [Full Text] [PDF]
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G. M. Passmore, A. A. Selyanko, M. Mistry, M. Al-Qatari, S. J. Marsh, E. A. Matthews, A. H. Dickenson, T. A. Brown, S. A. Burbidge, M. Main, et al. KCNQ/M Currents in Sensory Neurons: Significance for Pain Therapy J. Neurosci., August 6, 2003; 23(18): 7227 - 7236. [Abstract] [Full Text] [PDF]
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 P. Fong, B. E. Argent, W. B. Guggino, and M. A. Gray Characterization of vectorial chloride transport pathways in the human pancreatic duct adenocarcinoma cell line HPAF Am J Physiol Cell Physiol, August 1, 2003; 285(2): C433 - C445. [Abstract] [Full Text] [PDF]
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N. Gamper and M. S. Shapiro Calmodulin Mediates Ca2+-dependent Modulation of M-type K+ Channels J. Gen. Physiol., June 30, 2003; 122(1): 17 - 31. [Abstract] [Full Text] [PDF]
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J. K. Hadley, G. M. Passmore, L. Tatulian, M. Al-Qatari, F. Ye, A. D. Wickenden, and D. A. Brown Stoichiometry of Expressed KCNQ2/KCNQ3 Potassium Channels and Subunit Composition of Native Ganglionic M Channels Deduced from Block by Tetraethylammonium J. Neurosci., June 15, 2003; 23(12): 5012 - 5019. [Abstract] [Full Text] [PDF]
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 S. Ohya, G. P. Sergeant, I. A. Greenwood, and B. Horowitz Molecular Variants of KCNQ Channels Expressed in Murine Portal Vein Myocytes: A Role in Delayed Rectifier Current Circ. Res., May 16, 2003; 92(9): 1016 - 1023. [Abstract] [Full Text] [PDF]
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L Tatulian and D A Brown Effect of the KCNQ potassium channel opener retigabine on single KCNQ2/3 channels expressed in CHO cells J. Physiol., May 15, 2003; 549(1): 57 - 63. [Abstract] [Full Text] [PDF]
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D. Oliver, M. Knipper, C. Derst, and B. Fakler Resting Potential and Submembrane Calcium Concentration of Inner Hair Cells in the Isolated Mouse Cochlea Are Set by KCNQ-Type Potassium Channels J. Neurosci., March 15, 2003; 23(6): 2141 - 2149. [Abstract] [Full Text] [PDF]
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N. Gamper, J. D. Stockand, and M. S. Shapiro Subunit-Specific Modulation of KCNQ Potassium Channels by Src Tyrosine Kinase J. Neurosci., January 1, 2003; 23(1): 84 - 95. [Abstract] [Full Text] [PDF]
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H. Hu, K. Vervaeke, and J. F Storm Two forms of electrical resonance at theta frequencies, generated by M-current, h-current and persistent Na+ current in rat hippocampal pyramidal cells J. Physiol., December 15, 2002; 545(3): 783 - 805. [Abstract] [Full Text] [PDF]
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M M Shah, M Mistry, S J Marsh, D A Brown, and P Delmas Molecular correlates of the M-current in cultured rat hippocampal neurons J. Physiol., October 1, 2002; 544(1): 29 - 37. [Abstract] [Full Text] [PDF]
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H. Wen and I. B. Levitan Calmodulin Is an Auxiliary Subunit of KCNQ2/3 Potassium Channels J. Neurosci., September 15, 2002; 22(18): 7991 - 8001. [Abstract] [Full Text] [PDF]
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A. Alaburda, J.-F. Perrier, and J. Hounsgaard An M-like outward current regulates the excitability of spinal motoneurones in the adult turtle J. Physiol., May 1, 2002; 540(3): 875 - 881. [Abstract] [Full Text] [PDF]
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E. C. Cooper, E. Harrington, Y. N. Jan, and L. Y. Jan M Channel KCNQ2 Subunits Are Localized to Key Sites for Control of Neuronal Network Oscillations and Synchronization in Mouse Brain J. Neurosci., December 15, 2001; 21(24): 9529 - 9540. [Abstract] [Full Text] [PDF]
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K. Dedek, B. Kunath, C. Kananura, U. Reuner, T. J. Jentsch, and O. K. Steinlein Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K+ channel PNAS, September 19, 2001; (2001) 211431298. [Abstract] [Full Text] [PDF]
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M. J. Saganich, E. Machado, and B. Rudy Differential Expression of Genes Encoding Subthreshold-Operating Voltage-Gated K+ Channels in Brain J. Neurosci., July 1, 2001; 21(13): 4609 - 4624. [Abstract] [Full Text] [PDF]
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A A Selyanko, J K Hadley, and D A Brown Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells J. Physiol., July 1, 2001; 534(1): 15 - 24. [Abstract] [Full Text] [PDF]
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 C.-C. Shieh, M. Coghlan, J. P. Sullivan, and M. Gopalakrishnan Potassium Channels: Molecular Defects, Diseases, and Therapeutic Opportunities Pharmacol. Rev., December 1, 2000; 52(4): 557 - 594. [Abstract] [Full Text] [PDF]
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K. Dedek, B. Kunath, C. Kananura, U. Reuner, T. J. Jentsch, and O. K. Steinlein Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K+ channel PNAS, October 9, 2001; 98(21): 12272 - 12277. [Abstract] [Full Text] [PDF]
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P. Castaldo, E. M. del Giudice, G. Coppola, A. Pascotto, L. Annunziato, and M. Taglialatela Benign Familial Neonatal Convulsions Caused by Altered Gating of KCNQ2/KCNQ3 Potassium Channels J. Neurosci., January 15, 2002; 22(2): RC199 - RC199. [Abstract] [Full Text] [PDF]
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A. A. Selyanko, P. Delmas, J. K. Hadley, L. Tatulian, I. C. Wood, M. Mistry, B. London, and D. A. Brown Dominant-Negative Subunits Reveal Potassium Channel Families That Contribute to M-Like Potassium Currents J. Neurosci., March 1, 2002; 22(5): RC212 - RC212. [Abstract] [Full Text] [PDF]
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A. Alaburda, J.-F. Perrier, and J. Hounsgaard An M-like outward current regulates the excitability of spinal motoneurons in the adult turtle J. Physiol., March 15, 2002; (2002) 2001015982. [Abstract] [PDF]
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