Lack of potassium current in W309R mutant KCNQ3 channel causing benign familial neonatal convulsions (BFNC).
ABSTRACT BFNC is an autosomal dominant epileptic disorder caused by mutations of KCNQ2 or KCNQ3 potassium channel gene. W309R missense mutation in KCNQ3 gene was previously reported in a family with BFNC. In this study, potassium currents were recorded from HEK293 cells expressing both W309R mutant KCNQ3 and wild type KCNQ2 channels. We found a lack of potassium current in W309R mutant KCNQ3 and KCNQ2 channels, which can explain the hyper-excitability of CNS in patients with BFNC.
- SourceAvailable from: Francesco Miceli
Article: KCNQ3-Related Disorders[Show abstract] [Hide abstract]
ABSTRACT: KCNQ3-related disorders discussed in this GeneReview are benign familial neonatal epilepsy (BFNE) and benign familial infantile epilepsy (BFIE), seizure disorders that occur in children who have normal psychomotor development. In BFNE seizures begin in an otherwise healthy infant between days two and eight of life and spontaneously disappear between the first and the sixth to 12th month of life. Seizures are generally brief, lasting one to two minutes. Seizure types include tonic or apneic episodes, focal clonic activity, and autonomic changes. Motor activity may be confined to one body part, migrate to other regions, or generalize. Infants are well between seizures and feed normally. In BFIE seizures begin about age six months (range 3-8 months) and disappear about age two years. Seizures are generally brief, lasting two minutes; they appear as daily repeated clusters. Seizure type is usually focal, but can be also generalized, causing diffuse hypertonia with jerks of the limbs, head deviation, or motor arrest with unconsciousness and cyanosis.
- [Show abstract] [Hide abstract]
ABSTRACT: Mutations in the KCNQ2 and KCNQ3 genes encoding for Kv 7.2 (KCNQ2; Q2) and Kv 7.3 (KCNQ3; Q3) voltage-dependent K(+) channel subunits, respectively, cause neonatal epilepsies with wide phenotypic heterogeneity. In addition to benign familial neonatal epilepsy (BFNE), KCNQ2 mutations have been recently found in families with one or more family members with a severe outcome, including drug-resistant seizures with psychomotor retardation, EEG suppression-burst pattern (Ohtahara syndrome) and distinct neuroradiological features, a condition that was named "KCNQ2 encephalopathy". In the present paper, we describe clinical, genetic and functional data from 17 patients/families whose electro-clinical presentation was consistent with the diagnosis of BFNE. Sixteen different heterozygous mutations were found in KCNQ2, including 10 substitutions, three ins/del and three large deletions. One substitution was found in KCNQ3. Most of these mutations were novel, except for four KCNQ2 substitutions that were shown to be recurrent. Electrophysiological studies in mammalian cells revealed that homomeric or heteromeric KCNQ2 and/or KCNQ3 channels carrying mutant subunits with newly-found substitutions displayed reduced current densities. In addition, we describe, for the first time, that some mutations impair channel regulation by syntaxin-1A, highlighting a novel pathogenetic mechanism for KCNQ2-related epilepsies. This article is protected by copyright. All rights reserved.Human Mutation 12/2013; · 5.21 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Potassium channels participate in many critical biological functions and play important roles in a variety of diseases. In recent years, many significant discoveries have been made which motivate us to review these achievements. The focus of our review is mainly on three aspects. Firstly, we try to summarize the latest developments in structure determinants and regulation mechanism of all types of potassium channels. Secondly, we review some diseases induced by or related to these channels. Thirdly, both qualitative and quantitative approaches are utilized to analyze structural features of modulators of potassium channels. Our analyses further prove that modulators possess some certain natural product scaffolds. And pharmacokinetics parameters are important properties for organic molecules. Besides, with in silico methods, some features which can be used to differentiate modulators are derived. There is no doubt that all these studies on potassium channels as possible pharmaceutical targets will facilitate future translational research. All the strategies developed in this review could be extended to studies on other ion channels and proteins as well. This article is protected by copyright. All rights reserved.Chemical Biology & Drug Design 10/2013; · 2.47 Impact Factor
Epilepsy Research (2009) 84, 82—85
journal homepage: www.elsevier.com/locate/epilepsyres
Lack of potassium current in W309R mutant KCNQ3
channel causing benign familial neonatal
Yoshihiro Sugiuraa,∗, Fubito Nakatsub, Kiwamu Hiroyasuc, Atsushi Ishiid,
Shinichi Hirosed, Motohiro Okadae, Itsuki Jibikic, Hiroshi Ohnob,
Sunao Kanekof, Yoshikazu Ugawaa
aDepartment of Neurology, Fukushima Medical University, School of Medicine, Fukushima, Japan
bLaboratory for Epithelial Immunobiology, Research Center for Allergy and Immunology, RIKEN, Kanagawa, Japan
cDepartment of Neuropsychiatry, Kanazawa Medical University, Ishikawa, Japan
dDepartment of Pediatrics, Fukuoka University, School of Medicine, Fukuoka, Japan
eDepartment of Psychiatry, Graduate School of Medicine Division of Neuroscience, Mie University, Mie, Japan
fDepartment of Neuropsychiatry, Hirosaki University, School of Medicine, Aomori, Japan
Received 12 May 2008; received in revised form 7 November 2008; accepted 13 December 2008
Available online 23 January 2009
or KCNQ3 potassium channel gene. W309R missense mutation in KCNQ3 gene was previously
reported in a family with BFNC. In this study, potassium currents were recorded from HEK293
cells expressing both W309R mutant KCNQ3 and wild type KCNQ2 channels. We found a lack
of potassium current in W309R mutant KCNQ3 and KCNQ2 channels, which can explain the
hyper-excitability of CNS in patients with BFNC.
© 2008 Elsevier B.V. All rights reserved.
BFNC is an autosomal dominant epileptic disorder caused by mutations of KCNQ2
Benign familial neonatal convulsions (BFNC) is an autosomal
dominantly inherited disorder with benign course (Rett
and Teubel, 1964). The tonic-clonic convulsions develop
between day 3 after birth and 6 months of age and usually
∗Corresponding author at: Department of Neurology, Fukushima
Medical University, School of Medicine, 1 Hikarigaoka, Fukushima
960-1295, Japan. Tel.: +81 24 547 1247; fax: +81 24 548 3797.
E-mail address: firstname.lastname@example.org (Y. Sugiura).
remit spontaneously. Mutations of two KQT-like voltage-
gated potassium channel genes, KCNQ2 and KCNQ3, have
been reported in families with BFNC (Biervert et al., 1998;
Charlier et al., 1998). KCNQ2 and KCNQ3 subunits form a
heterotetramer as M-channel regulating repetitive neuronal
excitation in CNS (Schroeder et al., 1998). Functional
abnormalities of some mutant KCNQ2 subunits have been
physiologically studied (Biervert et al., 1998; Schroeder
et al., 1998), whereas only one report is available on
physiological properties of mutant KCNQ3 subunit of BFNC
(Singh et al., 2003). Most of KCNQ2 gene mutations caused
a depolarizing change of the voltage dependency or a mild
0920-1211/$ — see front matter © 2008 Elsevier B.V. All rights reserved.
Lack of K+current in W309R mutant KCNQ3 channel causing BFNC83
reduction of the maximal current carried by KCNQ2/KCNQ3
In a previous report, a novel missense mutation of KCNQ3
(c.925T→C) was identified in a Japanese family with BFNC
(Hirose et al., 2000). The transition substituted Tryptophan
in a pore domain (P-loop) of KCNQ3 with Arginine (W309R).
In this study, we investigate the electrophysiological prop-
erties of W309R mutant KCNQ3 subunit (KCNQ3(W309R)) to
elucidate the pathophysiology for BFNC.
The mouse KCNQ2 full-length cDNA was prepared by the recom-
binant PCR strategy (Nakatsu et al., 2000) using the EST clone
E530004J01 (Genbank accession number; BB609377) (Carninci et
al., 2005) that contained a 36-bp internal deletion, as a template.
The 5?half and the 3?half of the mouse KCNQ2 sequences were
amplified with the sense primer #398 (5?-CGCGTCGACCGCCATGGT-
GCAGAAGTCGCGCAAC-3?) and the anti-sense primer #452 (5?-
3?), and the sense primer #453 (5?-GTCAGTTTGAAAGATCGTGTCTTC-
TCCAGCCCCCGAGGCATGGCTGCCA-3?) and the anti-sense primer
tively. Finally, full-length mouse KCNQ2 cDNA was amplified using
the primers #398 and #399. Likewise, the mouse KCNQ3 full-length
cDNA was prepared by the PCR with the EST clone B630001K07 (Gen-
a 99-bp internal deletion as a template, and the following primers;
sense primer #396 (5?-AGCTAGCCGTGGGTCTCAAGGCTCGCACG-
3?) and anti-sense primer #455 (5?-TTCTGAGACTTCTTATGTTTT-
GGTACTTTATC-3?) for 5?half, and sense primer #456 (5?-AAAACATAA-
AGGAATGAACCATATGTAGCCAG-3?) and anti-sense primer #397 (5?-
GCTCGAGTTAAGTGGGCTTGTTGGAAGG-3?) for 3?half. Site-directed
mutagenesis was performed by the recombinant PCR method to
introduce the W309R mutation in the mouse KCNQ3 cDNA using the
sense primer (5?-AGATGCTCTGCGGTGGGGCCT-3?) and the antisense
primer (5?-AGGCCCCACCGCAGAGCATCT-3?). Bicistronic expression
vectors were constructed by introducing the full-length cDNAs into
the pIRES vector (Invitrogen).
Expression in HEK293 cells and electrophysiology
or pIRES-mKCNQ3(W309R)-mKCNQ2(WT) into the human cell line
HEK293 using PolyFect (Qiagen) (WT; wild type). 36—40h after
transfection, potassium currents were recorded from HEK293 cells
at room temperature (20—22◦C) by the whole cell patch clamp
technique with a pipette resistance of 1—3.5M?. The patch
pipettes were filled with 120 KCl, 5.37 CaCl2, 1.75 MgCl2, 1.6
ATP, 10 EGTA, and 10 HEPES, adjusted with KOH to pH 7.2 (in
mM). The bath solution contained 140 NaCl, 4 KCl, 2 CaCl2, 1
MgCl2, and 10 HEPES adjusted with NaOH to pH 7.4 (in mM). Cur-
rents were recorded in response to 600ms pulses applied every
10s in the range of −80mV to +40mV from a holding poten-
tial of −80mV with a patch clamp amplifier (AXOPATCH 200B)
and pCLAMP v.9.2 (Axon Instruments, Foster City, CA). The mean
(+SD) conductance—voltage (G/V) curve was obtained with Boltz-
600ms and holding potential is −80mV. A family of potassium currents recorded from KCNQ3(WT)/KCNQ2(WT) (A). The potas-
sium current did not appear in KCNQ3(W309R)/KCNQ2(WT) channel (B). Conductance—voltage curve (G/V curve) of wild type
KCNQ3/KCNQ2 current (C). Peak currents at +20mV in KCNQ3(WT)/KCNQ2(WT) channel (n=7) and in KCNQ3(W309R)/KCNQ2(WT)
channel (n=5) (D).
Potassium currents recorded from a KCNQ3/KCNQ2 channel expressed in HEK293 cells. The duration of step pulses is
84Y. Sugiura et al.
mann fitting by ORIGIN v.6.1 software (OriginLab, Northampton,
HEK293 cells with pIRES-mKCNQ3(W309R)-mKCNQ2(WT) were cul-
tured on glass coverslips for two days after transfection, and
immunostained as described elsewhere(Shah et al., 2002). The pri-
mary antibody was anti-KCNQ3 antibody (1:100; N-19 from Santa
Fig. 1 shows superimposed two sets of potassium cur-
rents recorded at room temperature (20—22◦C) from
HEK293 cells on which either KCNQ3(WT)/KCNQ2(WT)
expressed. Normal potassium currents were recorded
Conductance—voltage curve (G/V curve) for wild type
KCNQ3/KCNQ2 currents was obtained by normalizing the
tail currents to the maximal value recorded during repo-
larization to −80mV (Fig. 1C). The membrane potential
at 50% of the maximal current (V1/2) was −54.7mV for
significant currents (Fig. 1B). Peak current at +20mV
was 0.77±0.16nA in a wild type KCNQ3/KCNQ2 channel
(n=7) and 0.04±0.03nA in KCNQ3(W309R)/KCNQ2(WT)
channel (n=5) (Fig. 1D). A significant difference was
test, p<0.01). In addition, no current was induced in
KCNQ3(W309R)/KCNQ2(WT) channels at 36◦C (data not
Because potassium current did not appear in W309R
mutant channel, KCNQ3 immuno-staining was carried
out to cytologically confirm an expression of W309R
mutant KCNQ3/KCNQ2 channel on HEK293 cells. Anti-KCNQ3
against KCNQ3 chanel. KCNQ3 channels were expressed in
HEK293 cells into which W309R mutant KCNQ3/KCNQ2 gene
was transfected (A). KCNQ3 channel did not appear in a
negative control specimen (HEK293 cells) (B).
Immuno-cytochemical stainingwith antibody
immuno-staining labeled both the cytoplasm and the cell
surface of HEK293 cells into which W309R mutant KCNQ3
and KCNQ2 genes were transfected (Fig. 2).
We have shown that potassium currents were not evoked in
W309R mutant KCNQ3 with wild type KCNQ2 channels, which
were found in a family with BFNC, although the mutant
channels were expressed in transfected HEK293 cells. These
results indicate that this mutant channel does not function
at all. KCNQ2 and KCNQ3 subunits coassemble in a tetramer,
which result in large voltage dependent potassium cur-
rent (M-current). M-current repolarizes neuronal membrane
potential after a depolarization, thus limiting repetitive fir-
ing and causing spike-frequency adaptation (Wang et al.,
1998). Therefore, reduction of M-currents due to the abnor-
mal KCNQ2 or KCNQ3 channels results in hyper-excitability
of neurons. These may explain convulsions in patients with
W309R mutation of KCNQ3 channel.
The functional abnormalities of mutations for BFNC have
been mostly reported on KCNQ2 gene (Dedek et al., 2001;
Singh et al., 2003; Borgatti et al., 2004; Hunter et al.,
2006; Soldovieri et al., 2007). These reported mutant KCNQ2
and KCNQ3 channels caused depolarizing shift of the volt-
age dependency of M-current activation or reduction of the
M-current amplitude. In contrast, W309R mutant KCNQ3
channel studied here revealed no potassium conductance
at all. This mutation substituted hydrophobic amino acid
(Tryptophan) in a pore domain of KCNQ3 with basic amino
acid (Arginine), and may cause a conformation change of
the pore region in KCNQ2/KCNQ3 channel. This may result
in complete block of the pores.
Recently, heteromeric KCNQ3(W309R)/KCNQ2(WT) chan-
nels have been reported to reduce M-current amplitude
(Uehara et al., 2008), however our results showed the com-
plete block of M-channel. A possible explanation for the
discrepancy between our results and others may be as fol-
lows. We used pIRES vector which is a mammalian expression
vector to express two genes by cloning them into two mul-
tiple cloning sites located on either side of the internal
ribosome entry site (IRES) because KCNQ2 and KCNQ3 chan-
nel express one by one (Martinez Salas, 1999). Therefore,
in our preparation, all channels have mutant components in
a population. However, in other studies, KCNQ2 and KCNQ3
channels were expressed by co-transfection of each KCNQ2
and KCNQ3 gene, and a part of homomeric channels may
have no mutations in a population. These normal channels
must cause some amount of potassium currents recorded
and cause small currents in the preparation.
We revealed the lack of M-current caused by the mis-
sense mutation of KCNQ3 gene, which was found in a family
with BFNC. BFNC is characterized by the age-dependent
development and spontaneous remission of convulsions
because age-dependent reduction of inhibitory KCNQ chan-
nel activity and age-dependent functional switching to
the GABAergic-system in neonatal CNS (Okada et al.,
2003). However, delayed psychomotor development or men-
tal retardation was reported recently in a part of BFNC
cases (Steinlein et al., 2007). In addition, one recent
report showed that M-channel blockade severely acceler-
Lack of K+current in W309R mutant KCNQ3 channel causing BFNC85
ated hypoxia induced neurodegeneration (Gamper et al.,
2006). Consequently, it is unclear whether an effect of the
complete block of M-channel is transient in BFNC. However,
this issue may be solved after similar approaches to many
types of mutations in the future.
Our research was conducted as part of a comprehensive
project organized by The Epilepsy Genetic Study Group,
Japan (Chairperson, S. K.). Author Y.U. has received support
from Research Project Grants-in-aid for Scientific Research
No. 16500194 from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan, grants for the
Research Committee on rTMS treatment of movement dis-
orders from the Ministry of Health, Labour and Welfare of
Japan (17231401), the Research Committee on Dystonia, the
Ministry of Health, Labour and Welfare of Japan, grants
from the Committee of the Study of Human Exposure to
EMF, Ministry of Internal Affairs and Communications, a grant
from Fukushima Medical University Research Project. None
of the authors has any conflict of interest to disclose. The
authors thank the FANTOM Consortium and RIKEN Genome
Exploration Research Group and Genome Science Group for
generous gift of the EST clones.
Biervert, C., Schroeder, B.C., Kubisch, C., Berkovic, S.F., Prop-
ping, P., Jentsch, T.J., Steinlein, O.K., 1998. A potassium
channel mutation in neonatal human epilepsy. Science 279,
Borgatti, R., Zucca, C., Cavallini, A., Ferrario, M., Panzeri, C.,
Castaldo, P., Soldovieri, M.V., Baschirotto, C., Bresolin, N., Dalla
Bernardina, B., Taglialatela, M., Bassi, M.T., 2004. A novel muta-
tion in KCNQ2 associated with BFNC, drug resistant epilepsy, and
mental retardation. Neurology 63, 57—65.
Carninci, P., Kasukawa, T., Katayama, S., Gough, J., Frith, M.C.,
et al., 2005. The transcriptional landscape of the mammalian
genome. Science 309, 1559—1563.
Charlier, C., Singh, N.A., Ryan, S.G., Lewis, T.B., Reus, B.E., Leach,
R.J., Leppert, M., 1998. A pore mutation in a novel KQT-like
potassium channel gene in an idiopathic epilepsy family. Nat.
Genet. 18, 53—55.
Dedek, K., Kunath, B., Kananura, C., Reuner, U., Jentsch, T.J.,
Steinlein, O.K., 2001. Myokymia and neonatal epilepsy caused
by a mutation in the voltage sensor of the KCNQ2K+channel.
Proc. Natl. Acad. Sci. U.S.A. 98, 12272—12277.
Gamper, N., Zaika, O., Li, Y., Martin, P., Hernandez, C.C., Perez,
M.R., Wang, A.Y., Jaffe, D.B., Shapiro, M.S., 2006. Oxidative
modification of M-type K+channels as a mechanism of cytopro-
tective neuronal silencing. EMBO J. 25, 4996—5004.
Hirose, S., Zenri, F., Akiyoshi, H., Fukuma, G., Iwata, H., Inoue, T.,
Yonetani, M., Tsutsumi, M., Muranaka, H., Kurokawa, T., Hanai,
T., Wada, K., Kaneko, S., Mitsudome, A., 2000. A novel mutation
of KCNQ3 (c.925T–>C) in a Japanese family with benign familial
neonatal convulsions. Ann. Neurol. 47, 822—826.
Hunter, J., Maljevic, S., Shankar, A., Siegel, A., Weissman, B., Holt,
P., Olson, L., Lerche, H., Escayg, A., 2006. Subthreshold changes
epilepsy. Neurobiol. Dis. 24, 194—201.
Martinez Salas, E., 1999. Internal ribosome entry site biology and its
use in expression vectors. Curr. Opin. Biotechnol. 10, 458—464.
Nakatsu, F., Sakuma, M., Matsuo, Y., Arase, H., Yamasaki, S., Naka-
mura, N., Saito, T., Ohno, H., 2000. A Di-leucine signal in
the ubiquitin moiety. Possible involvement in ubiquitination-
mediated endocytosis. J. Biol. Chem. 275, 26213—26219.
Okada, M., Zhu, G., Hirose, S., Ito, K.I., Murakami, T., Wakui, M.,
Kaneko, S., 2003. Age-dependent modulation of hippocampal
excitability by KCNQ-channels. Epilepsy Res. 53, 81—94.
Rett, A., Teubel, R., 1964. Neugeborenen Krampfe im Rahmen
einer epileptisch belasten Familie. Wien. Klin. Wochenschr. 76,
Schroeder, B.C., Kubisch, C., Stein, V., Jentsch, T.J., 1998. Moder-
ate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3K+
channels causes epilepsy. Nature 396, 687—690.
Shah, M.M., Mistry, M., Marsh, S.J., Brown, D.A., Delmas, P., 2002.
Molecular correlates of the M-current in cultured rat hippocam-
pal neurons. J. Physiol. 544, 29—37.
Singh, N.A., Westenskow, P., Charlier, C., Pappas, C., Leslie, J., Dil-
lon, J., Anderson, V.E., Sanguinetti, M.C., Leppert, M.F., 2003.
KCNQ2 and KCNQ3 potassium channel genes in benign familial
neonatal convulsions: expansion of the functional and mutation
spectrum. Brain 126, 2726—2737.
Soldovieri, M.V., Cilio, M.R., Miceli, F., Bellini, G., Miraglia del Giu-
dice, E., Castaldo, P., Hernandez, C.C., Shapiro, M.S., Pascotto,
A., Annunziato, L., Taglialatela, M., 2007. Atypical gating of M-
type potassium channels conferred by mutations in uncharged
residues in the S4 region of KCNQ2 causing benign familial neona-
tal convulsions. J. Neurosci. 27, 4919—4928.
Steinlein, O.K., Conrad, C., Weidner, B., 2007. Benign familial
neonatal convulsions: always benign? Epilepsy Res. 73, 245—249.
Uehara, A., Nakamura, Y., Shioya, T., Hirose, S., Yasukochi, M.,
Uehara, K., 2008. Altered KCNQ3 potassium channel function
caused by the W309R pore-helix mutation found in human
epilepsy. J. Membr. Biol. 222, 55—63.
Wang, H.S., Pan, Z., Shi, W., Brown, B.S., Wymore, R.S., Cohen,
I.S., Dixon, J.E., McKinnon, D., 1998. KCNQ2 and KCNQ3 potas-
sium channel subunits: molecular correlates of the M-channel.
Science 282, 1890—1893.