G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
potassium voltage-gated channel, KQT-like subfamily, member 2
G00000090 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000033049 (Vega human gene)
ENSG00000075043 (Ensembl human gene)
3785 (Entrez Gene)
427 (G2Cdb plasticity & disease)
KCNQ2 (GeneCards)
602235 (OMIM)
Marker Symbol
HGNC:6296 (HGNC)
Protein Sequence
O43526 (UniProt)

Synonyms (5)

  • BFNC
  • ENB1
  • KCNA11
  • Kv7.2

Literature (73)

Pubmed - other

  • Novel mutation in KCNQ2 causing benign familial neonatal seizures.

    Goldberg-Stern H, Kaufmann R, Kivity S, Afawi Z and Heron SE

    Department of Child Neurology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel. hagoldberg@clalit.org.il

    Potassium channel subunits encoded by several genes of the KCNQ family underlie the M-current. Specifically, KCNQ2 and KCNQ3 play a major role at most neuronal sites. Mutations in KCNQ2 or KCNQ3 that reduce the M-current are responsible for benign familial neonatal seizures, a rare autosomal dominant idiopathic epilepsy of the newborn. The aim of this study was to investigate a single family with benign familial neonatal seizures for mutations in KCNQ genes and to analyze the association of mutation type with disease prognosis. A family in which members in several generations had signs and symptoms compatible with a diagnosis of benign familial neonatal seizures had DNA testing with single-stranded conformation polymorphism analysis for various mutations known to cause benign familial neonatal seizures. A novel KCNQ2 mutation c.63-66delGGTG (p.K21fsX40), causing a framework shift and early chain termination, was identified in the affected family members. In all cases, there was complete remission of the seizures after the neonatal period. This KCNQ2 mutation has implications for diagnosis and prognosis of familial neonatal seizures. Its presence suggests a benign disease with good prognosis and its identification can spare patients and physicians the need for extensive investigations or prolonged therapy.

    Pediatric neurology 2009;41;5;367-70

  • Functional analysis of novel KCNQ2 mutations found in patients with Benign Familial Neonatal Convulsions.

    Volkers L, Rook MB, Das JH, Verbeek NE, Groenewegen WA, van Kempen MJ, Lindhout D and Koeleman BP

    Complex Genetics Section, DBG-Department of Medical Genetics, University Medical Center Utrecht, The Netherlands.

    Benign Familial Neonatal Convulsions (BFNC) are a rare epilepsy disorder with an autosomal-dominant inheritance. It is linked to mutations in the potassium channel genes KCNQ2 and KCNQ3. These encode for Kv7.2 and Kv7.3 potassium ion channels, which produce an M-current that regulates the potential firing action in neurons through modulation of the membrane potential. We report on the biophysical and biochemical properties of V589X, T359K and P410fs12X mutant-KCNQ2 ion channels that were detected in three BFNC families. Mutant KCNQ2 cDNAs were co-expressed with WT-KCNQ2 and KCNQ3 cDNAs in HEK293 cells to mimic heterozygous expression of the KCNQ2 mutations in BFNC patients. The resulting potassium currents were measured using patch-clamp techniques and showed an approximately 75% reduction in current and a depolarized shift in the voltage dependence of activation. Furthermore, the time-constant of activation of M-currents in cells expressing T359K and P410fs12X was slower compared to cells expressing only wild-type proteins. Immunofluorescent labeling of HEK293 cells stably expressing GFP-tagged KCNQ2-WT or mutant alpha-subunits indicated cell surface expression of WT, V589X and T359K mutants, suggesting a loss-of-function, while P410fs12X was predominantly retained in the ER and sub-cellular compartments outside the ER suggesting an effectively haplo-insufficient effect.

    Neuroscience letters 2009;462;1;24-9

  • Sodium and potassium channel dysfunctions in rare and common idiopathic epilepsy syndromes.

    Hahn A and Neubauer BA

    Department of Neuropediatrics, Feulgenstr. 12, D-35385 Giessen, Germany.

    Mutations in the SCN1A gene are found in up to 80% of individuals with severe myoclonic epilepsy of infancy (SMEI), and mutations in KCNQ2 and KCNQ3 were identified in benign familial neonatal convulsions (BFNC) as well as in single families with Rolandic epilepsy (RE) and idiopathic generalized epilepsies (IGE). This paper summarizes recent findings concerning sodium (SCN1A) and potassium channel (KCNQ2 and KCNQ3) dysfunctions in the pathogenesis of rare and common idiopathic epilepsies (IE). SMEI, severe idiopathic generalized epilepsy of infancy (SIGEI), and myoclonic-astatic epilepsy (MAE) are rare IE. Because of some semeiologic overlap, a comparative analysis of the SCN1A gene performed in 20 patients with MAE and in 18 with SIGEI. This revealed mutations in three subjects with SIGEI only. Since BFNC are over-represented in families with RE, a mutational analysis was performed in 58 families with RE with and without BNFC. This revealed functionally relevant mutations in two index cases with BNFC, and three missense mutations (one resulting in a significantly reduced potassium current amplitude) in three patients with RE, but without BNFC. One KCNQ3 missense variant was also detected in eight out of 455 IGE patients but not in 454 controls, and a silent KCNQ2-SNP was found over-represented in both epilepsy samples. These findings confirm that mutations in the SCN1A gene are mainly involved in the pathogenesis of SMEI, rarely in that of SIGEI, and are commonly not found in patients with MAE. They also demonstrate that sequence variations of the KCNQ2 and KCNQ3 genes may contribute to the etiology of common IE syndromes.

    Brain & development 2009;31;7;515-20

  • Calmodulin activation limits the rate of KCNQ2 K+ channel exit from the endoplasmic reticulum.

    Alaimo A, Gómez-Posada JC, Aivar P, Etxeberría A, Rodriguez-Alfaro JA, Areso P and Villarroel A

    Unidad de Biofísica, Consejo Superior de Investigaciones Cientificas-Universidad del Pais Vasco/Euskal Herriko Unibersitatea (CSIC-UPV/EHU), Spain.

    The potential regulation of protein trafficking by calmodulin (CaM) is a novel concept that remains to be substantiated. We proposed that KCNQ2 K+ channel trafficking is regulated by CaM binding to the C-terminal A and B helices. Here we show that the L339R mutation in helix A, which is linked to human benign neonatal convulsions, perturbs CaM binding to KCNQ2 channels and prevents their correct trafficking to the plasma membrane. We used glutathione S-transferase fused to helices A and B to examine the impact of this and other mutations in helix A (I340A, I340E, A343D, and R353G) on the interaction with CaM. The process appears to require at least two steps; the first involves the transient association of CaM with KCNQ2, and in the second, the complex adopts an "active" conformation that is more stable and is that which confers the capacity to exit the endoplasmic reticulum. Significantly, the mutations that we have analyzed mainly affect the stability of the active configuration of the complex, whereas Ca2+ alone appears to affect the initial binding step. The spectrum of responses from this collection of mutants revealed a strong correlation between adopting the active conformation and channel trafficking in mammalian cells. These data are entirely consistent with the concept that CaM bound to KCNQ2 acts as a Ca2+ sensor, conferring Ca2+ dependence to the trafficking of the channel to the plasma membrane and fully explaining the requirement of CaM binding for KCNQ2 function.

    The Journal of biological chemistry 2009;284;31;20668-75

  • KCNQ2 and KCNQ3 mutations contribute to different idiopathic epilepsy syndromes.

    Neubauer BA, Waldegger S, Heinzinger J, Hahn A, Kurlemann G, Fiedler B, Eberhard F, Muhle H, Stephani U, Garkisch S, Eeg-Olofsson O, Müller U and Sander T

    Department of Pediatric Neurology, University of Giessen-Marburg, Feulgenstrasse 12, D-35385 Giessen, Germany. bernd.a.neubauer@paediat.med.uni-giessen.de

    Objective: To explore the involvement of M-type potassium channels KCNQ2, Q3, and Q5 in the pathogenesis of common idiopathic epilepsies.

    Methods: Sequence analysis of the KCNQ2, Q3, and Q5 coding regions was performed in a screening sample consisting of 58 nuclear families with rolandic epilepsy. Subsequently, an association study was conducted for all discovered variants in a case-control sample comprising 459 German patients with idiopathic generalized epilepsy (IGE) and 462 population controls.

    Results: An in-frame deletion of codon 116 in KCNQ2 (p.Lys116del) and a missense mutation in KCNQ3 (p.Glu299Lys) were detected in two index cases exhibiting rolandic epilepsy and benign neonatal convulsions. Both mutations resulted in reduced potassium current amplitude in Xenopus oocytes. Mutation analysis of families with rolandic epilepsy without neonatal seizures discovered three novel missense variations (KCNQ2 p.Ile592Met, KCNQ3 p.Ala381Val, KCNQ3 p.Pro574Ser). The KCNQ2 p.Ile592Met variant displayed a significant reduction of potassium current amplitude in Xenopus oocytes and was present only once in 552 controls. Both missense variants identified in KCNQ3 (p.Ala381Val and p.Pro574Ser) were present in all affected family members and did not occur in controls, but did not show obvious functional abnormalities. The KCNQ3 missense variant p.Pro574Ser was also detected in 8 of 455 IGE patients but not in 454 controls (p = 0.008). In KCNQ2, a silent single nucleotide polymorphism (rs1801545) was found overrepresented in both epilepsy samples (IGE, p = 0.004).

    Conclusion: Sequence variations of the KCNQ2 and KCNQ3 genes may contribute to the etiology of common idiopathic epilepsy syndromes.

    Neurology 2008;71;3;177-83

  • A schizophrenia-linked mutation in PIP5K2A fails to activate neuronal M channels.

    Fedorenko O, Strutz-Seebohm N, Henrion U, Ureche ON, Lang F, Seebohm G and Lang UE

    Department of Physiology, University of Tuebingen, Gmelinstr. 5, 72076, Tuebingen, Germany.

    Rationale: Evidence for an association between phosphatidylinositol-4-phosphate 5-kinase II alpha (PIP5K2A) and schizophrenia was recently obtained and replicated in several samples. PIP5K2A controls the function of KCNQ channels via phosphatidylinositol-4,5-bisphosphate (PIP2) synthesis. Interestingly, recent data suggest that KCNQ channels suppress basal activity of dopaminergic neurons and dopaminergic firing. Activation of KCNQ accordingly attenuates the central stimulating effects of dopamine, cocaine, methylphenidate, and phenylcyclidine.

    Objective: The aim of this study was to explore the functional relevance of PIP5K2A, which might influence schizophrenic behavior.

    Here, we study the effects of the neuronal PIP5K2A on KCNQ2, KCNQ5, KCNQ2/KCNQ3, and KCNQ3/KCNQ5 in the Xenopus expression system.

    Results: We find that wild-type PIP5K2A but not the schizophrenia-associated mutant (N251S)-PIP5K2A activates heteromeric KCNQ2/KCNQ3 and KCNQ3/KCNQ5, the molecular correlate of neuronal M channels. Homomeric KCNQ2 and KCNQ5 channels were not activated by the kinase indicating that the presence of KCNQ3 in the channel complex is required for the kinase-mediated effects. Acute application of PI(4,5)P2 and a PIP2 scavenger indicates that the mutation N251S renders the kinase PIP5K2A inactive.

    Conclusions: Our results suggest that the schizophrenia-linked mutation of the kinase results in reduced KCNQ channel function and thereby might explain the loss of dopaminergic control in schizophrenic patients. Moreover, the addictive potential of dopaminergic drugs often observed in schizophrenic patients might be explained by this mechanism. At least, the insufficiency of (N251S)-PIP5K2A to stimulate neuronal M channels may contribute to the clinical phenotype of schizophrenia.

    Psychopharmacology 2008;199;1;47-54

  • Phosphatidylinositol 4,5-bisphosphate hydrolysis mediates histamine-induced KCNQ/M current inhibition.

    Liu B, Liang H, Liu L and Zhang H

    Department of Pharmacology, Hebei Medical University, Shijiazhuang, Hebei Province, China.

    The M-type potassium channel, of which its molecular basis is constituted by KCNQ2-5 homo- or heteromultimers, plays a key role in regulating neuronal excitability and is modulated by many G protein-coupled receptors. In this study, we demonstrate that histamine inhibits KCNQ2/Q3 currents in human embryonic kidney (HEK)293 cells via phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolysis mediated by stimulation of H(1) receptor and phospholipase C (PLC). Histamine inhibited KCNQ2/Q3 currents in HEK293 cells coexpressing H(1) receptor, and this effect was totally abolished by H(1) receptor antagonist mepyramine but not altered by H(2) receptor antagonist cimetidine. The inhibition of KCNQ currents was significantly attenuated by a PLC inhibitor U-73122 but not affected by depletion of internal Ca(2+) stores or intracellular Ca(2+) concentration ([Ca(2+)](i)) buffering via pipette dialyzing BAPTA. Moreover, histamine also concentration dependently inhibited M current in rat superior cervical ganglion (SCG) neurons by a similar mechanism. The inhibitory effect of histamine on KCNQ2/Q3 currents was entirely reversible but became irreversible when the resynthesis of PIP(2) was impaired with phosphatidylinsitol-4-kinase inhibitors. Histamine was capable of producing a reversible translocation of the PIP(2) fluorescence probe PLC(delta1)-PH-GFP from membrane to cytosol in HEK293 cells by activation of H(1) receptor and PLC. We concluded that the inhibition of KCNQ/M currents by histamine in HEK293 cells and SCG neurons is due to the consumption of membrane PIP(2) by PLC.

    American journal of physiology. Cell physiology 2008;295;1;C81-91

  • Developmental changes in KCNQ2 and KCNQ3 expression in human brain: possible contribution to the age-dependent etiology of benign familial neonatal convulsions.

    Kanaumi T, Takashima S, Iwasaki H, Itoh M, Mitsudome A and Hirose S

    Department of Pediatrics, School of Medicine, Fukuoka University, 45-1, 7-chome Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. kanaumi@minf.med.fukuoka-u.ac.jp

    Several mutations of KCNQ2 and KCNQ3 are considered to be associated with benign familial neonatal convulsions (BFNC). BFNC is characterized by seizures starting within several days of life and spontaneous remission within weeks to months. KCNQ channel is a heteromeric voltage-dependent potassium channel consisting of KCNQ2 and KCNQ3 subunits. To clarify the age-dependent etiology of BFNC, we examined the developmental changes in KCNQ2 and KCNQ3 expression in human hippocampus, temporal lobe, cerebellum and medulla oblongata obtained from 23 subjects who died at 22 gestation weeks to adulthood. Formalin-fixed and paraffin-embedded specimens were used for immunohistochemistry. Unique developmental changes in KCNQ2 and KCNQ3 were found in each region. A high expression of KCNQ2 was identified in the hippocampus, temporal cortex, cerebellar cortex and medulla oblongata in fetal life, but such expression decreased after birth. The expression of KCNQ3 increased in late fetal life to infancy. Simultaneous and high expressions of KCNQ2 and KCNQ3 were observed in each region from late fetal life to early infancy, coinciding with the time when BFNC occurs. Such coexpression may contribute to the pathogenesis of BFNC.

    Brain & development 2008;30;5;362-9

  • Calmodulin regulates the trafficking of KCNQ2 potassium channels.

    Etxeberria A, Aivar P, Rodriguez-Alfaro JA, Alaimo A, Villacé P, Gómez-Posada JC, Areso P and Villarroel A

    Unidad de Biofísica, CSIC-UPV/EHU, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain.

    Voltage-dependent potassium KCNQ2 (Kv7.2) channels play a prominent role in the control of neuronal excitability. These channels must associate with calmodulin to function correctly and, indeed, a mutation (R353G) that impairs this association provokes the onset of a form of human neonatal epilepsy known as benign familial neonatal convulsions (BFNC). We show here that perturbation of calmodulin binding leads to endoplasmic reticulum (ER) retention of KCNQ2, reducing the number of channels that reach the plasma membrane. Interestingly, elevating the expression of calmodulin in the BFNC mutant partially restores the intracellular distribution of the KCNQ channel. In contrast, overexpression of a Ca(2+)-binding incompetent calmodulin or sequestering of calmodulin promotes the retention of wild-type channels in the ER. Thus, a direct interaction with Ca(2+)-calmodulin appears to be critical for the correct activity of KCNQ2 potassium channels as it controls the channels' exit from the ER.

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;4;1135-43

  • Germ-line mutation of KCNQ2, p.R213W, in a Japanese family with benign familial neonatal convulsion.

    Sadewa AH, Sasongko TH, Gunadi, Lee MJ, Daikoku K, Yamamoto A, Yamasaki T, Tanaka S, Matsuo M and Nishio H

    Department of Public Health, Kobe University Graduate School of Medicine, Kobe, Japan.

    Background: Benign familial neonatal convulsion (BFNC) is an autosomal-dominantly inherited epilepsy of neonates. The KCNQ2 and KCNQ3 genes have been cloned as the responsible genes for BFNC. Detection of mutations in these genes is helpful for confirmation of BFNC or differential diagnosis of convulsive disorders in the neonatal period.

    Methods: A Japanese family with BFNC was investigated. Two siblings were clinically diagnosed as having BFNC. KCNQ2 and KCNQ3 were screened for mutations using a combination of polymerase chain reaction and denaturing high-performance liquid chromatography. Nucleotide substitutions were confirmed by direct sequencing.

    Results: In the affected siblings a C-to-T heterozygous substitution was detected at nucleotide 683 (c.683C>T) in KCNQ2, leading to substitution of arginine with tryptophan at amino acid position 213 (p.R213W) in the S4 voltage-sensing domain of the KCNQ2 protein. The detected mutation may disrupt this highly conserved region among potassium channel proteins. The c.683C>T substitution in KCNQ2 was not present in the parents. KCNQ3 was also analyzed and a single nucleotide polymorphism, c.1241A>G (National Center for Biotechnology Information (NCBI), SNP ID: rs2303995), was detected in the index family.

    Conclusions: Two siblings with BFNC had a novel heterozygous missense mutation, p.R213W, in KCNQ2. This mutation may affect potassium gating, leading to neuronal excitability or convulsions in the patients. Furthermore, neither of the parents had the p.R213W mutation, indicating that it was a germ-line mutation. The possibility of recurrence of such a germ-line mutation in the next siblings should be explained during genetic counseling.

    Pediatrics international : official journal of the Japan Pediatric Society 2008;50;2;167-71

  • N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243): a novel, selective KCNQ2/Q3 potassium channel activator.

    Wickenden AD, Krajewski JL, London B, Wagoner PK, Wilson WA, Clark S, Roeloffs R, McNaughton-Smith G and Rigdon GC

    Icagen, Inc., 4222 Emperor Blvd., Durham, NC 27703, USA. jkrajewski@icagen.com

    KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) are voltage-gated K(+) channel subunits that underlie the neuronal M current. In humans, mutations in these genes lead to a rare form of neonatal epilepsy (Biervert et al., 1998; Singh et al., 1998), suggesting that KCNQ2/Q3 channels may be attractive targets for novel antiepileptic drugs. In the present study, we have identified the compound N-(6-chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243) as a selective activator of the neuronal M current and KCNQ2/Q3 channels. In SH-SY5Y human neuroblastoma cells, ICA-27243 produced membrane potential hyperpolarization that could be prevented by coadministration with the M-current inhibitors 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone dihydrochloride (XE-991) and linopirdine. ICA-27243 enhanced both (86)Rb(+) efflux (EC(50) = 0.2 microM) and whole-cell currents in Chinese hamster ovary cells stably expressing heteromultimeric KCNQ2/Q3 channels (EC(50) = 0.4 microM). Activation of KCNQ2/Q3 channels was associated with a hyperpolarizing shift of the voltage dependence of channel activation (V((1/2)) shift of -19 mV at 10 microM). In contrast, ICA-27243 was less effective at activating KCNQ4 and KCNQ3/Q5 and was selective over a wide range of neurotransmitter receptors and ion channels such as voltage-dependent sodium channels and GABA-gated chloride channels. ICA-27243 (1-10 microM) was found to reversibly suppress seizure-like activity in an ex vivo hippocampal slice model of epilepsy and demonstrated in vivo anticonvulsant activity (ED(50) = 8.4 mg/kg) in the mouse maximal electroshock epilepsy model. In conclusion, ICA-27243 represents the first member of a novel chemical class of selective KCNQ2/Q3 activators with anticonvulsant-like activity in experimental models of epilepsy.

    Molecular pharmacology 2008;73;3;977-86

  • Neutralization of a negative charge in the S1-S2 region of the KV7.2 (KCNQ2) channel affects voltage-dependent activation in neonatal epilepsy.

    Wuttke TV, Penzien J, Fauler M, Seebohm G, Lehmann-Horn F, Lerche H and Jurkat-Rott K

    Neurological Clinic, University of Ulm, Germany.

    The voltage-gated potassium channels KV7.2 and KV7.3 (genes KCNQ2 and KCNQ3) constitute a major component of the M-current controlling the firing rate in many neurons. Mutations within these two channel subunits cause benign familial neonatal convulsions (BFNC). Here we identified a novel BFNC-causing mutation (E119G) in the S1-S2 region of KV7.2. Electrophysiological investigations in Xenopus oocytes using two-microelectrode voltage clamping revealed that the steady-state activation curves for E119G alone and its coexpressions with KV7.2 and/or KV7.3 wild-type (WT) channels were significantly shifted in the depolarizing direction compared to KV7.2 or KV7.2/KV7.3. These shifts reduced the relative current amplitudes for mutant channels particularly in the subthreshold range of an action potential (about 45% reduction at --50 mV for E119G compared to KV7.2, and 33% for E119G/KV7.3 compared to KV7.2/KV7.3 channels). Activation kinetics were significantly slowed for mutant channels. Our results indicate that small changes in channel gating at subthreshold voltages are sufficient to cause neonatal seizures and demonstrate the importance of the M-current for this voltage range. This was confirmed by a computer model predicting an increased burst duration for the mutation. On a molecular level, these results reveal a critical role in voltage sensing of the negatively charged E119 in S1-S2 of KV7.2, a region that-- according to molecular modelling - might interact with a positive charge in the S4 segment.

    The Journal of physiology 2008;586;2;545-55

  • Deletions or duplications in KCNQ2 can cause benign familial neonatal seizures.

    Heron SE, Cox K, Grinton BE, Zuberi SM, Kivity S, Afawi Z, Straussberg R, Berkovic SF, Scheffer IE and Mulley JC

    Background: Benign familial neonatal seizures are most often caused by mutations in the voltage-gated potassium channel subunit gene KCNQ2. More than 60 mutations have been described in BFNS families, approximately half of which lead to protein truncation. The hypothesis of this study was that deletion or duplication of >or=1 exons of KCNQ2 could cause BFNS in cases without coding or splicing mutations.

    Methods: Multiplex ligation-dependent probe amplification (MLPA) was used to test a group of 21 unrelated patients with clinical features consistent with either BFNS, benign familial neonatal-infantile seizures or sporadic neonatal seizures, for exonic deletions and duplications.

    Results: Three deletions and one duplication mutation were identified in four familial cases and cascade testing of their available family members showed that the mutations segregated with the phenotype in each family. The junction fragment for one of the deletions was amplified by PCR and sequenced to characterise the breakpoint and verify that a deletion had occurred.

    Conclusions: Submicroscopic deletions or duplications of KCNQ2 are seen in a significant proportion of BFNS families: four of nine (44%) cases previously testing negative for coding or splice site mutation by sequencing KCNQ2 and KCNQ3. MLPA is an efficient second-tier testing strategy for KCNQ2 to identify pathogenic intragenic mutations not detectable by conventional DNA sequencing methods.

    Journal of medical genetics 2007;44;12;791-6

  • Peripheral nerve hyperexcitability due to dominant-negative KCNQ2 mutations.

    Wuttke TV, Jurkat-Rott K, Paulus W, Garncarek M, Lehmann-Horn F and Lerche H

    Institut für Angewandte Physiologie, Universität Ulm, Germany.

    Background: Peripheral nerve hyperexcitability (PNH) is characterized by muscle overactivity due to spontaneous discharges of lower motor neurons usually associated with antibodies against voltage-gated potassium channels. PNH may also occur in combination with episodic ataxia or epilepsy caused by mutations in K(V)1.1 or K(V)7.2 channels. Only one PNH-associated mutation has been described so far in K(V)7.2 (R207W), in a family with both PNH and neonatal seizures.

    Methods: PNH was characterized by video and electromyography. The KCNQ2 gene was sequenced and K(V)7.2 channels were functionally characterized using two-microelectrode voltage-clamping in Xenopus oocytes.

    Results: In a patient with PNH without other neurologic symptoms, we identified a novel KCNQ2 mutation predicting loss of a charged residue within the voltage sensor of K(V)7.2 (R207Q). Functional analysis of both PNH-associated mutants revealed large depolarizing shifts of the conductance-voltage relationships and marked slowing of the activation time course compared to wild type (WT) channels, less pronounced for R207Q than R207W. Co-expression of both mutant with WT channels revealed a dominant negative effect reducing the relative current amplitudes after short depolarizations by >70%. The anticonvulsant retigabine, an activator of neuronal K(V)7 channels, reversed the depolarizing shift.

    Conclusions: Mutations in KCNQ2 can cause idiopathic PNH alone and should be considered in sporadic cases. Both K(V)7.2 mutants produce PNH by changing voltage-dependent activation with a dominant negative effect on the WT channel. This distinguishes them from all hitherto examined Kv7.2 or K(V)7.3 mutations which cause neonatal seizures by haploinsufficiency. Retigabine may be beneficial in treating PNH.

    Neurology 2007;69;22;2045-53

  • A novel missense mutation (N258S) in the KCNQ2 gene in a Turkish family afflicted with benign familial neonatal convulsions (BFNC).

    Yalçin O, Cağlayan SH, Saltik S, Cokar O, Ağan K, Dervent A and Steinlein OK

    Department of Molecular Biology and Genetics, Boğaziçi University, Istanbul, Turkey.

    Benign familial neonatal convulsions (BFNC) is a rare monogenic subtype of idiopathic epilepsy exhibiting autosomal dominant mode of inheritance. The disease is caused by mutations in the two homologous genes KCNQ2 and KCNQ3 that encode the subunits of the voltage-gated potassium channel. Most KCNQ2 mutations are found in the pore region and the cytoplasmic C domain. These mutations are either deletions/insertions that result in frameshift or truncation of the protein product, splice-site variants or missense mutations. This study reveals a novel missense mutation (N258S) in the KCNQ2 gene between the S5 domain and the pore of the potassium channel in two BFNC patients in a Turkish family. The absence of the mutation both in the healthy members of the family and in a control group, and the lack of any other change in the KCNQ2 gene of the patients indicate that N258S substitution is a pathogenic mutation leading to epileptic seizures in this family.

    The Turkish journal of pediatrics 2007;49;4;385-9

  • Electrostatic interaction of internal Mg2+ with membrane PIP2 Seen with KCNQ K+ channels.

    Suh BC and Hille B

    Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195, USA.

    Activity of KCNQ (Kv7) channels requires binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) from the plasma membrane. We give evidence that Mg(2+) and polyamines weaken the KCNQ channel-phospholipid interaction. Lowering internal Mg(2+) augmented inward and outward KCNQ currents symmetrically, and raising Mg(2+) reduced currents symmetrically. Polyvalent organic cations added to the pipette solution had similar effects. Their potency sequence followed the number of positive charges: putrescine (+2) < spermidine (+3) < spermine (+4) < neomycin (+6) < polylysine (>+6). The inhibitory effects of Mg(2+) were reversible with sequential whole-cell patching. Internal tetraethylammonium ion (TEA) gave classical voltage-dependent block of the pore with changes of the time course of K(+) currents. The effect of polyvalent cations was simpler, symmetric, and without changes of current time course. Overexpression of phosphatidylinositol 4-phosphate 5-kinase Igamma to accelerate synthesis of PIP(2) attenuated the sensitivity to polyvalent cations. We suggest that Mg(2+) and other polycations reduce the currents by electrostatic binding to the negative charges of PIP(2), competitively reducing the amount of free PIP(2) available for interaction with channels. The dose-response curves could be modeled by a competition model that reduces the pool of free PIP(2). This mechanism is likely to modulate many other PIP(2)-dependent ion channels and cellular processes.

    Funded by: NINDS NIH HHS: NS08174, R01 NS008174, R37 NS008174

    The Journal of general physiology 2007;130;3;241-56

  • Atypical gating of M-type potassium channels conferred by mutations in uncharged residues in the S4 region of KCNQ2 causing benign familial neonatal convulsions.

    Soldovieri MV, Cilio MR, Miceli F, Bellini G, Miraglia del Giudice E, Castaldo P, Hernandez CC, Shapiro MS, Pascotto A, Annunziato L and Taglialatela M

    Section of Pharmacology, Department of Neuroscience, University of Naples Federico II, 80131 Naples, Italy.

    Heteromeric assembly of KCNQ2 and KCNQ3 subunits underlie the M-current (I(KM)), a slowly activating and noninactivating neuronal K(+) current. Mutations in KCNQ2 and KCNQ3 genes cause benign familial neonatal convulsions (BFNCs), a rare autosomal-dominant epilepsy of the newborn. In the present study, we describe the identification of a novel KCNQ2 heterozygous mutation (c587t) in a BFNC-affected family, leading to an alanine to valine substitution at amino acid position 196 located at the N-terminal end of the voltage-sensing S(4) domain. The consequences on KCNQ2 subunit function prompted by the A196V substitution, as well as by the A196V/L197P mutation previously described in another BFNC-affected family, were investigated by macroscopic and single-channel current measurements in CHO cells transiently transfected with wild-type and mutant subunits. When compared with KCNQ2 channels, homomeric KCNQ2 A196V or A196V/L197P channels showed a 20 mV rightward shift in their activation voltage dependence, with no concomitant change in maximal open probability or single-channel conductance. Furthermore, current activation kinetics of KCNQ2 A196V channels displayed an unusual dependence on the conditioning prepulse voltage, being markedly slower when preceded by prepulses to more depolarized potentials. Heteromeric channels formed by KCNQ2 A196V and KCNQ3 subunits displayed gating changes similar to those of KCNQ2 A196V homomeric channels. Collectively, these results reveal a novel role for noncharged residues in the N-terminal end of S(4) in controlling gating of I(KM) and suggest that gating changes caused by mutations at these residues may decrease I(KM) function, thus causing neuronal hyperexcitability, ultimately leading to neonatal convulsions.

    Funded by: NINDS NIH HHS: R01 NS043394; Telethon: TI_GGP030209

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2007;27;18;4919-28

  • Self-assembly of the isolated KCNQ2 subunit interaction domain.

    Wehling C, Beimgraben C, Gelhaus C, Friedrich T, Saftig P, Grötzinger J and Schwake M

    Institute of Biochemistry, Christian-Albrechts-University Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany.

    Mutations in the KCNQ2 gene cause myokymia and neonatal epilepsy, indicating that this K(+) channel regulates the excitability of lower motoneurons and CNS neurons. Little is known about the parameters that direct the assembly of this multimeric molecule and other KCNQ subunits. Here, we show that the carboxy-terminal subunit interaction domain of KCNQ2 autonomously folds and assembles into tetramers. This domain contains a bipartite coiled-coil motif. Whereas structural integrity of the second coiled-coil motif is crucial for tetramer formation, that of the first motif is less important. These data suggest a crucial role of coiled-coil motifs in tetrameric KCNQ channel assembly.

    FEBS letters 2007;581;8;1594-8

  • Rapid chemically induced changes of PtdIns(4,5)P2 gate KCNQ ion channels.

    Suh BC, Inoue T, Meyer T and Hille B

    Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195, USA.

    To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

    Funded by: NIAMS NIH HHS: AR17803, R01 AR017803; NIGMS NIH HHS: GM63702, R01 GM030179, R01 GM030179-24A1, R01 GM030179-25, R01 GM063702; NIMH NIH HHS: MH64801, R01 MH064801; NINDS NIH HHS: NS08174, R01 NS008174, R37 NS008174

    Science (New York, N.Y.) 2006;314;5804;1454-7

  • Does diacylglycerol regulate KCNQ channels?

    Suh BC and Hille B

    Department of Physiology and Biophysics, University of Washington School of Medicine, G-424 Health Sciences Building, P.O. Box 357290, Seattle, WA, 98195-7290, USA.

    Some ion channels are regulated by inositol phospholipids and by the products of cleavage by phospholipase C (PLC). KCNQ channels (Kv7) require membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) and are turned off when muscarinic receptors stimulate cleavage of PIP(2) by PLC. We test whether diacylglycerols are also important in the regulation of KCNQ2/KCNQ3 channels using electrophysiology and fluorescent translocation probes as indicators for PIP(2) and diacylglycerol in tsA cells. The cells are transfected with M(1) muscarinic receptors, channel subunits, and translocation probes. Although they cause translocation of a fluorescent probe with a diacylglycerol-binding C1 domain, exogenously applied diacylglycerol (oleoyl-acetyl-glycerol and dioctanoyl glycerol) and phorbol ester do not mimic or occlude the suppression of KCNQ current by muscarinic agonist. Blocking the metabolism of endogenous diacylglycerol by inhibiting diacylglycerol kinase with R59022 or R59949 slows the decay of diacylglycerol twofold but does not mimic or occlude muscarinic regulation and recovery of current. Blocking diacylglycerol lipase with RHC-80267 also does not occlude muscarinic modulation of current. We conclude that the diacylglycerol produced during activation of PLC, any activation of protein kinase C that it may stimulate, and downstream products of its metabolism are not essential players in the acute muscarinic modulation of KCNQ channels.

    Funded by: NINDS NIH HHS: NS08174

    Pflugers Archiv : European journal of physiology 2006;453;3;293-301

  • Infantile seizures and other epileptic phenotypes in a Chinese family with a missense mutation of KCNQ2.

    Zhou X, Ma A, Liu X, Huang C, Zhang Y, Shi R, Mao S, Geng T and Li S

    Department of Pediatrics, First Affiliated Hospital, Ion Channel Disease Laboratory, Medical College of Xi'an Jiaotong University, Xi'an, Shaanxi, People's Republic of China. zhouxih@mail.xjtu.edu.cn

    Introduction: Benign familial infantile seizures (BFIS) is a form of idiopathic epilepsy characterized by clusters of afebrile seizures occurring around the sixth month of life and a favorable outcome. Linkage analysis has revealed that three chromosomal segments, 19q12-q13.1, 16p12-q12, and 2q23-31, are linked to this disorder.

    We report here a large Chinese family in which all 17 affected members had had infantile seizures with onset at age 2-4 months, with two of these also manifesting seizures later in life accompanied with either choreoathetosis or myokymia. Linkage analysis in this family confirmed a previous report of genetic heterogeneity in BFIS - since linkage was excluded at the above-mentioned known BFIS loci - and suggested a possible linkage to the KCNQ2 gene, which is believed to be a voltage gated potassium channel gene responsible for benign familial neonatal seizures (BFNS).

    Sequencing of the KCNQ2 gene revealed that all 17 affected family members carried a heterozygous Gly-to-Val (G271V) mutation in the conserved pore region that resulted from a guanine-to-thymine transition in exon 5 of KCNQ2. The same mutation with a comparable localization in the KCNQ3 (G310V) gene has been found in BFNS patients. The same conserved amino acid was also found to be mutated in the KCNQ1 gene in a family with Long QT Syndrome.

    European journal of pediatrics 2006;165;10;691-5

  • Severe epilepsy resulting from genetic interaction between Scn2a and Kcnq2.

    Kearney JA, Yang Y, Beyer B, Bergren SK, Claes L, Dejonghe P and Frankel WN

    Department of Human Genetics, 4909 Buhl Building 0618, 1241 E. Catherine Street, Ann Arbor, MI 48109-0618, USA. jkearney@umich.edu

    A mutation in the voltage-gated sodium-channel Scn2a results in moderate epilepsy in transgenic Scn2a(Q54) mice maintained on a C57BL/6J strain background. The onset of progressive epilepsy begins in adults with short-duration partial seizures that originate in the hippocampus. The underlying abnormality is an increase in persistent sodium current in hippocampal neurons. The voltage-gated potassium channel Kcnq2 is responsible for generating M current (I(KM)) that is thought to control excitability and limit repetitive firing of hippocampal neurons. To determine whether impaired M current would exacerbate the seizure phenotype of Scn2a(Q54) mice, we carried out genetic crosses with two mutant alleles of Kcnq2. Szt1 mice carry a spontaneous deletion that removes the C-terminal domain of Kcnq2. A novel Kcnq2 missense mutation V182M was identified by screening the offspring of ENU-treated males for reduced threshold to electrically evoked minimal clonic seizures. Double mutant mice carrying the Scn2a(Q54) transgene together with either of the Kcnq2 mutations exhibited severe epilepsy with early onset, generalized tonic-clonic seizures and juvenile lethality by 3 weeks of age. This dramatic exacerbation of the sodium-channel mutant phenotype indicates that M current plays a critical role in preventing seizure initiation and spreading in this animal model. The genetic interaction between Scn2a and Kcnq2 demonstrates that combinations of mild alleles of monogenic epilepsy genes can result in severe disease and provides a model for complex inheritance of human epilepsy. The data suggest that interaction between these genes might contribute to the variable expressivity observed in human families with sodium-channel mutations. In a screen of 23 SMEI patients with missense mutations of SCN1A, no second-site mutations in KCNQ2 were identified.

    Funded by: NINDS NIH HHS: R21 NS046315, U01 NS41215

    Human molecular genetics 2006;15;6;1043-8

  • Decreased subunit stability as a novel mechanism for potassium current impairment by a KCNQ2 C terminus mutation causing benign familial neonatal convulsions.

    Soldovieri MV, Castaldo P, Iodice L, Miceli F, Barrese V, Bellini G, Miraglia del Giudice E, Pascotto A, Bonatti S, Annunziato L and Taglialatela M

    Division of Pharmacology, Department of Neuroscience, University of Naples Federico II, 80131 Naples.

    KCNQ2 and KCNQ3 K+ channel subunits underlie the muscarinic-regulated K+ current (I(KM)), a widespread regulator of neuronal excitability. Mutations in KCNQ2- or KCNQ3-encoding genes cause benign familiar neonatal convulsions (BFNCs), a rare autosomal-dominant idiopathic epilepsy of the newborn. In the present study, we have investigated, by means of electrophysiological, biochemical, and immunocytochemical techniques in transiently transfected cells, the consequences prompted by a BFNC-causing 1-bp deletion (2043deltaT) in the KCNQ2 gene; this frameshift mutation caused the substitution of the last 163 amino acids of the KCNQ2 C terminus and the extension of the subunit by additional 56 residues. The 2043deltaT mutation abolished voltage-gated K+ currents produced upon homomeric expression of KCNQ2 subunits, dramatically reduced the steady-state cellular levels of KCNQ2 subunits, and prevented their delivery to the plasma membrane. Metabolic labeling experiments revealed that mutant KCNQ2 subunits underwent faster degradation; 10-h treatment with the proteasomal inhibitor MG132 (20 microm) at least partially reversed such enhanced degradation. Co-expression with KCNQ3 subunits reduced the degradation rate of mutant KCNQ2 subunits and led to their expression on the plasma membrane. Finally, co-expression of KCNQ2 2043deltaT together with KCNQ3 subunits generated functional voltage-gated K+ currents having pharmacological and biophysical properties of heteromeric channels. Collectively, the present results suggest that mutation-induced reduced stability of KCNQ2 subunits may cause epilepsy in neonates.

    Funded by: Telethon: GGP030209

    The Journal of biological chemistry 2006;281;1;418-28

  • Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels.

    Surti TS, Huang L, Jan YN, Jan LY and Cooper EC

    Graduate Group in Biophysics, University of California, 1550 4th Street, Room 484, San Francisco, CA 94143-0725, USA.

    Neuronal potassium channel subunits of the KCNQ (Kv7) family underlie M-current (I(M)), and may also underlie the slow potassium current at the node of Ranvier, I(Ks). I(M) and I(Ks) are outwardly rectifying currents that regulate excitability of neurons and myelinated axons, respectively. Studies of native I(M) and heterologously expressed Kv7 subunits suggest that, in vivo, KCNQ channels exist within heterogeneous, multicomponent protein complexes. KCNQ channel properties are regulated by protein phosphorylation, protein-protein interactions, and protein-lipid interactions within such complexes. To better understand the regulation of neuronal KCNQ channels, we searched directly for posttranslational modifications on KCNQ2/KCNQ3 channels in vivo by using mass spectrometry. Here we describe two sites of phosphorylation. One site, specific for KCNQ3, appears functionally silent in electrophysiological assays but is located in a domain previously shown to be important for subunit tetramerization. Mutagenesis and electrophysiological studies of the second site, located in the S4-S5 intracellular loop of all KCNQ subunits, reveal a mechanism of channel inhibition.

    Funded by: NCRR NIH HHS: P41 RR001614, RR01614, RR14606; NIMH NIH HHS: MH65334, R01 MH065334; NINDS NIH HHS: R21 NS042100, R21 NS42100

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;49;17828-33

  • Functional analysis of novel KCNQ2 and KCNQ3 gene variants found in a large pedigree with benign familial neonatal convulsions (BFNC).

    Bassi MT, Balottin U, Panzeri C, Piccinelli P, Castaldo P, Barrese V, Soldovieri MV, Miceli F, Colombo M, Bresolin N, Borgatti R and Taglialatela M

    IRCCS E. Medea, Via D.L. Monza 20, 23842 Bosisio Parini Lecco, Italy. mtbassi@bp.lnf.it

    Benign familial neonatal convulsion (BFNC) is a rare autosomal dominant disorder caused by mutations in KCNQ2 and KCNQ3, two genes encoding for potassium channel subunits. A large family with nine members affected by BFNC is described in the present study. All affected members of this family carry a novel deletion/insertion mutation in the KCNQ2 gene (c.761_770del10insA), which determines a premature truncation of the protein. In addition, in the family of the proposita's father, a novel sequence variant (c.2687A>G) in KCNQ3 leading to the p.N821S amino acid change was detected. When heterologously expressed in Chinese hamster ovary cells, KCNQ2 subunits carrying the mutation failed to form functional potassium channels in homomeric configuration and did not affect channels formed by KCNQ2 and/or KCNQ3 subunits. On the other hand, homomeric and heteromeric potassium channels formed by KCNQ3 subunits carrying the p.N821S variant were indistinguishable from those formed by wild-type KCNQ3 subunits. Finally, the current density of the cells mimicking the double heterozygotic condition for both KCNQ2 and KCNQ3 alleles of the proband was decreased by approximately 25% when compared to cells expressing only wild-type alleles. Collectively, these results suggest that, in the family investigated, the KCNQ2 mutation is responsible for the BFNC phenotype, possibly because of haplo-insufficiency, whereas the KCNQ3 variant is functionally silent, a result compatible with its lack of segregation with the BFNC phenotype.

    Funded by: Telethon: GGP030209

    Neurogenetics 2005;6;4;185-93

  • International Union of Pharmacology. LIII. Nomenclature and molecular relationships of voltage-gated potassium channels.

    Gutman GA, Chandy KG, Grissmer S, Lazdunski M, McKinnon D, Pardo LA, Robertson GA, Rudy B, Sanguinetti MC, Stühmer W and Wang X

    Department of Microbiology and Molecular Genetics, University of California, Irvine, CA, USA. gagutman@uci.edu

    Pharmacological reviews 2005;57;4;473-508

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • De novo KCNQ2 mutations in patients with benign neonatal seizures.

    Claes LR, Ceulemans B, Audenaert D, Deprez L, Jansen A, Hasaerts D, Weckx S, Claeys KG, Del-Favero J, Van Broeckhoven C and De Jonghe P

    Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), University of Antwerp, Antwerpen, Belgium.

    Benign familial neonatal convulsions (BFNC) are characterized by unprovoked seizures during the first weeks of life with spontaneous remission after a few months. Mutations have been identified in the voltage-gated potassium ion channels KCNQ2 and KCNQ3. The authors performed a mutation analysis of KCNQ2 and KCNQ3 in six patients of whom four had no family history of neonatal seizures. The authors identified three KCNQ2 mutations in four patients that all arose de novo.

    Neurology 2004;63;11;2155-8

  • Dual phosphorylations underlie modulation of unitary KCNQ K(+) channels by Src tyrosine kinase.

    Li Y, Langlais P, Gamper N, Liu F and Shapiro MS

    Department of Physiology, University of Texas Health Science Center, San Antonio, Texas 78229, USA.

    Src tyrosine kinase suppresses KCNQ (M-type) K(+) channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed.

    Funded by: NIDDK NIH HHS: R01 DK52933; NINDS NIH HHS: R01 NS43394

    The Journal of biological chemistry 2004;279;44;45399-407

  • Three mechanisms underlie KCNQ2/3 heteromeric potassium M-channel potentiation.

    Etxeberria A, Santana-Castro I, Regalado MP, Aivar P and Villarroel A

    Instituto Cajal Consejo Superior de Investigaciones Científicas (CSIC), 28002 Madrid, Spain.

    The non-inactivating potassium M-current exerts a strong influence on neuronal excitability. The channels responsible for this current are made up of KCNQ subunits, and mutations in most of these produce human pathologies. Notably, in terms of excitation, mutations in either KCNQ2 or KCNQ3 lead to benign neonatal familial convulsions. Although a mere reduction of 25% in KCNQ2/3 function can increase excitability to epileptogenic levels, the potentiation of these subunits has anti-epileptogenic effects. After KCNQ2/3 heteromerization, current levels can augment as much as 10-fold, and we have discovered that there are three processes underlying this potentiation. First, there is an increase in the number of channels inserted in the membrane after heteromerization of the C-terminal region. Second, the N-terminal domain from KCNQ2 exerts a negative influence on the current level. Finally, Ala 315 of KCNQ3, a residue located in the inner vestibule after the selectivity filter, plays a critical role in preventing current flow in KCNQ3 homomeric channels, whereas it is permissive in heteromers in combination with Thr at the equivalent 276 position of KCNQ2.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;41;9146-52

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • A novel mutation in KCNQ2 associated with BFNC, drug resistant epilepsy, and mental retardation.

    Borgatti R, Zucca C, Cavallini A, Ferrario M, Panzeri C, Castaldo P, Soldovieri MV, Baschirotto C, Bresolin N, Dalla Bernardina B, Taglialatela M and Bassi MT

    Divisione di Neuroriabilitazione 1, IRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy. borgatti@bp.lnf.it

    Background: Benign familial neonatal convulsion (BFNC) is a rare autosomal dominant disorder caused by mutations in two genes, KCNQ2 and KCNQ3, encoding for potassium channel subunits underlying the M-current. This current limits neuronal hyperexcitability by causing spike-frequency adaptation.

    Methods: The authors describe a BFNC family with four affected members: two of them exhibit BFNC only while the other two, in addition to BFNC, present either with a severe epileptic encephalopathy or with focal seizures and mental retardation.

    Results: All affected members of this family carry a novel missense mutation in the KCNQ2 gene (K526N), disrupting the tri-dimensional conformation of a C-terminal region of the channel subunit involved in accessory protein binding. When heterologously expressed in CHO cells, potassium channels containing mutant subunits in homomeric or heteromeric configuration with wild-type KCNQ2 and KCNQ3 subunits exhibit an altered voltage-dependence of activation, without changes in intracellular trafficking and plasma membrane expression.

    Conclusion: The KCNQ2 K526N mutation may affect M-channel function by disrupting the complex biochemical signaling involving KCNQ2 C-terminus. Genetic rather than acquired factors may be involved in the pathophysiology of the phenotypic variability of the neurologic symptoms associated with BFNC in the described family.

    Funded by: Telethon: GGP030209

    Neurology 2004;63;1;57-65

  • A novel mutation in KCNQ2 gene causes benign familial neonatal convulsions in a Chinese family.

    Tang B, Li H, Xia K, Jiang H, Pan Q, Shen L, Long Z, Zhao G and Cai F

    Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, PR China. bstang7398@yahoo.com.cn

    Benign familial neonatal convulsions (BFNC) are a rare autosomal dominant inherited epilepsy syndrome. Two voltage-gated potassium channel genes, KCNQ2 on chromosome 20q13.3 and KCNQ3 on chromosome 8q24, have been identified as the genes responsible for benign familial neonatal convulsions. By linkage analysis and mutation analysis of KCNQ2 gene, we found a novel frameshift mutation of KCNQ2 gene, 1931delG, in a large Chinese family with benign familial neonatal convulsions. This mutation is located in the C-terminus of KCNQ2, in codon 644 predicting the replacement of the last 201 amino acids with a stretch of 257 amino acids showing a completely different sequence. An unusual clinical feature of this family is that the seizures of every patient did not remit until 12 to 18 months. This is the first report of KCNQ2 gene mutation in China.

    Journal of the neurological sciences 2004;221;1-2;31-4

  • Complete loss of the cytoplasmic carboxyl terminus of the KCNQ2 potassium channel: a novel mutation in a large Czech pedigree with benign neonatal convulsions or other epileptic phenotypes.

    Pereira S, Roll P, Krizova J, Genton P, Brazdil M, Kuba R, Cau P, Rektor I and Szepetowski P

    INSERM U491, Marseille, France.

    Purpose: Benign neonatal familial convulsions (BNFCs) represent a rare epileptic disorder with autosomal dominant mode of inheritance. To date, two voltage-gated potassium (K+) channel genes, KCNQ2 and KCNQ3, have been identified in typical BNFC families. The study of new pedigrees may help detect new mutations and define genotype-phenotype correlations.

    Methods: A large Czech family was detected in which BNFC was inherited together with a broad range of various nonneonatal epileptic phenotypes. Genetic linkage study and direct mutation analysis were performed to find the disease-causing mutation.

    Results: In seven patients with BNFCs and no recurrence of seizures, a novel two-base-pair deletion (1369del2) was identified within the coding sequence of the KCNQ2 gene. The mutation led to a putative protein that lacked nearly all its carboxyl terminus part, which plays a critical role for the accurate expression of the functional K+ channels. Three patients with generalized tonic-clonic seizures (GTCSs), all without any history of BNFCs, also displayed 1369del2. Three other patients with other idiopathic epileptic phenotypes did not have the mutation.

    Conclusions: A novel 2-bp deletion within the coding sequence of the potassium channel KCNQ2 gene was detected in patients from a large and heterogeneous family with BNFCs or non-BNFC seizures.

    Epilepsia 2004;45;4;384-90

  • Novel mutations in the KCNQ2 gene link epilepsy to a dysfunction of the KCNQ2-calmodulin interaction.

    Richards MC, Heron SE, Spendlove HE, Scheffer IE, Grinton B, Berkovic SF, Mulley JC and Davy A

    Department of Laboratory Genetics, Women's and Children's Hospital, North Adelaide, South Australia, Australia.

    Journal of medical genetics 2004;41;3;e35

  • An unappreciated role for RNA surveillance.

    Hillman RT, Green RE and Brenner SE

    Department of Bioengineering, University of California, Berkeley, CA 94720-3102, USA.

    Background: Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons (PTC+ mRNAs). In mammals, a termination codon is recognized as premature if it lies more than about 50 nucleotides upstream of the final intron position. More than a third of reliably inferred alternative splicing events in humans have been shown to result in PTC+ mRNA isoforms. As the mechanistic details of NMD have only recently been elucidated, we hypothesized that many PTC+ isoforms may have been cloned, characterized and deposited in the public databases, even though they would be targeted for degradation in vivo.

    Results: We analyzed the human alternative protein isoforms described in the SWISS-PROT database and found that 144 (5.8% of 2,483) isoform sequences amenable to analysis, from 107 (7.9% of 1,363) SWISS-PROT entries, derive from PTC+ mRNA.

    Conclusions: For several of the PTC+ isoforms we identified, existing experimental evidence can be reinterpreted and is consistent with the action of NMD to degrade the transcripts. Several genes with mRNA isoforms that we identified as PTC+--calpain-10, the CDC-like kinases (CLKs) and LARD--show how previous experimental results may be understood in light of NMD.

    Funded by: NHGRI NIH HHS: K22 HG000056, K22 HG00056, T32 HG000047, T32 HG00047

    Genome biology 2004;5;2;R8

  • KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum.

    Singh NA, Westenskow P, Charlier C, Pappas C, Leslie J, Dillon J, Anderson VE, Sanguinetti MC, Leppert MF and BFNC Physician Consortium

    Department of Human Genetics, University of Utah, Salt Lake City, Utah 84112, USA. nandas@genetics.utah.edu

    Benign familial neonatal convulsions (BFNC) is a rare autosomal dominant generalized epilepsy of the newborn infant. Seizures occur repeatedly in the first days of life and remit by approximately 4 months of age. Previously our laboratory cloned two novel potassium channel genes, KCNQ2 and KCNQ3, and showed that they are mutated in patients with BFNC. In this report, we characterize the breakpoints of a previously reported interstitial deletion in the KCNQ2 gene and show that only KCNQ2 is deleted. We identify 11 novel mutations in KCNQ2 and one novel mutation in the KCNQ3 potassium channel genes. In one family, the phenotype extends beyond neonatal seizures and includes rolandic seizures, and a subset of families has onset of seizures in infancy. In the Xenopus oocyte expression system, we characterize five KCNQ2 and one KCNQ3 disease-causing mutations. These mutations cause a variable loss of function, and selective effects on the biophysical properties of KCNQ2/KCNQ3 heteromultimeric channels. We report here the first dominant negative mutation in KCNQ2 that has a phenotype of neonatal seizures without permanent clinical CNS impairment.

    Funded by: NINDS NIH HHS: R01 NS-32666

    Brain : a journal of neurology 2003;126;Pt 12;2726-37

  • The secreted protein discovery initiative (SPDI), a large-scale effort to identify novel human secreted and transmembrane proteins: a bioinformatics assessment.

    Clark HF, Gurney AL, Abaya E, Baker K, Baldwin D, Brush J, Chen J, Chow B, Chui C, Crowley C, Currell B, Deuel B, Dowd P, Eaton D, Foster J, Grimaldi C, Gu Q, Hass PE, Heldens S, Huang A, Kim HS, Klimowski L, Jin Y, Johnson S, Lee J, Lewis L, Liao D, Mark M, Robbie E, Sanchez C, Schoenfeld J, Seshagiri S, Simmons L, Singh J, Smith V, Stinson J, Vagts A, Vandlen R, Watanabe C, Wieand D, Woods K, Xie MH, Yansura D, Yi S, Yu G, Yuan J, Zhang M, Zhang Z, Goddard A, Wood WI, Godowski P and Gray A

    Departments of Bioinformatics, Molecular Biology and Protein Chemistry, Genentech, Inc, South San Francisco, California 94080, USA. hclark@gene.com

    A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel sec 169a reted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

    Genome research 2003;13;10;2265-70

  • A novel KCNQ2 K+ channel mutation in benign neonatal convulsions and centrotemporal spikes.

    Coppola G, Castaldo P, Miraglia del Giudice E, Bellini G, Galasso F, Soldovieri MV, Anzalone L, Sferro C, Annunziato L, Pascotto A and Taglialatela M

    Department of Child Neuropsychiatry, 2nd University of Naples, Italy.

    Patients with benign familial neonatal convulsions (BFNC) may develop various epilepsies or epilepsy-associated EEG traits. A heterozygous 1-base pair deletion (2043DeltaT) in the KCNQ2 gene encoding for K+ channel subunits was found in a patient with BFNC who showed centrotemporal spikes at age 3 years. Electrophysiologic studies showed that mutant K+ channel subunits failed to give rise to functional homomeric channels or exert dominant-negative effects when expressed with KCNQ2/KCNQ3 subunits.

    Neurology 2003;61;1;131-4

  • Stoichiometry of expressed KCNQ2/KCNQ3 potassium channels and subunit composition of native ganglionic M channels deduced from block by tetraethylammonium.

    Hadley JK, Passmore GM, Tatulian L, Al-Qatari M, Ye F, Wickenden AD and Brown DA

    Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom.

    KCNQ2 and KCNQ3 potassium-channel subunits can form both homomeric and heteromeric channels; the latter are thought to constitute native ganglionic M channels. We have tried to deduce the stoichiometric contributions of KCNQ2 and KCNQ3 subunits to currents generated by the coexpression of KCNQ2 and KCNQ3 cDNA plasmids in Chinese hamster ovary (CHO) cells, and to native M currents in dissociated rat superior cervical ganglion (SCG) neurons, by comparing the block of these currents produced by tetraethylammonium (TEA) with the block of currents generated by a tandem KCNQ3/2 construct. TEA concentration-inhibition curves against coexpressed KCNQ2 plus KCNQ3 currents, and against native M currents in SCG neurons from 6-week-old [postnatal day 45 (P45)] rats, were indistinguishable from those for the expressed tandem construct, and fully accorded with a 1:1 stoichiometry. Inhibition curves in neurons from younger (P17) rats could be better fitted assuming an additional small proportion of current carried by KCNQ2 homomultimers. Single-cell PCR yielded signals for KCNQ2, KCNQ3, and KCNQ5 mRNAs in all SCG neurons tested from both P17 and P45 rats. Quantitative PCR of whole-ganglion mRNA revealed stable levels of KCNQ2 and KCNQ5 mRNA between P7 and P45, but excess and incrementing levels of KCNQ3 mRNA. Increasing levels of KCNQ3 protein between P17 and P45 were confirmed by immunocytochemistry. We conclude that coexpressed KCNQ2 plus KCNQ3 cDNAs generate channels with 1:1 (KCNQ2:KCNQ3) stoichiometry in CHO cells and that native M channels in SCG neurons adopt the same conformation during development, assisted by the increased expression of KCNQ3 mRNA and protein.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2003;23;12;5012-9

  • AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists.

    Hoshi N, Zhang JS, Omaki M, Takeuchi T, Yokoyama S, Wanaverbecq N, Langeberg LK, Yoneda Y, Scott JD, Brown DA and Higashida H

    Department of Biophysical Genetics, Kanazawa University Graduate School of Medicine, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8640, Japan. hoshin@ohsu.edu

    M-type (KCNQ2/3) potassium channels are suppressed by activation of G(q/11)-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(DeltaA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(DeltaA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.

    Funded by: NIGMS NIH HHS: GM48231, R01 GM048231, R37 GM048231

    Nature neuroscience 2003;6;6;564-71

  • C-terminal interaction of KCNQ2 and KCNQ3 K+ channels.

    Maljevic S, Lerche C, Seebohm G, Alekov AK, Busch AE and Lerche H

    Department of Applied Physiology, University of Ulm, Germany.

    Coexpression of KCNQ2 and KCNQ3 channels results in a 10-fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C-terminus as a potential interaction domain by construction of chimeras with interchanged C-termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C-terminus (Q1ctQ2) showed an 8-fold increase in current amplitude, and Q1ctQ3 a 3-fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C-terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C-terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C-terminus for a dominant-negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A-domain, plus either 40 residues downstream of the A-domain (B-domain) or the proximal C-terminus between S6 and the A-domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10-fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage-dependent activation. The proximal part of the KCNQ3 C-terminus was necessary to produce these effects. Our results indicate that specific parts of the C-terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C-terminus are important for regulating current amplitude.

    The Journal of physiology 2003;548;Pt 2;353-60

  • Neonatal convulsions and epileptic encephalopathy in an Italian family with a missense mutation in the fifth transmembrane region of KCNQ2.

    Dedek K, Fusco L, Teloy N and Steinlein OK

    Zentrum für Molekulare Neurobiologie (ZMNH), University Hamburg, Hamburg, Germany.

    Mutations in the voltage gated K(+)-channel gene KCNQ2 are known to cause benign familial neonatal convulsions (BFNC), which are characterized by a benign course, spontaneous remission and normal psychomotor development. Most KCNQ2 mutations can be predicted to truncate the protein. Only a few amino acid exchanges have been found, and their localization was restricted to either the pore region or the fourth or sixth transmembrane region (TM). We have now identified the first KCNQ2 mutation located within TM5, affecting a highly conserved serine in amino acid position 247 of the predicted protein. The clinical history of the two affected family members is not compatible with typical BFNC. The poor outcome in the index patient raises the question if at least some KCNQ2 mutations might increase the risk to develop therapy-resistant epilepsy. Additional studies are needed to evaluate the possibility of a causal relationship between KCNQ2 mutations and severe early infantile epilepsy.

    Epilepsy research 2003;54;1;21-7

  • A carboxy-terminal domain determines the subunit specificity of KCNQ K+ channel assembly.

    Schwake M, Jentsch TJ and Friedrich T

    Centre for Molecular Neurobiology Hamburg, ZMNH, Falkenried 94, Germany.

    Mutations in KCNQ K(+) channel genes underlie several human pathologies. KCNQ alpha-subunits form either homotetramers or hetero-oligomers with a restricted subset of other KCNQ alpha-subunits or with KCNE beta-subunits. KCNQ1 assembles with KCNE beta-subunits but not with other KCNQ alpha-subunits. By contrast, KCNQ3 interacts with KCNQ2, KCNQ4 and KCNQ5. Using a chimaeric strategy, we show that a cytoplasmic carboxy-terminal subunit interaction domain (sid) suffices to transfer assembly properties between KCNQ3 and KCNQ1. A chimaera (KCNQ1-sid(Q3)) carrying the si domain of KCNQ3 within the KCNQ1 backbone interacted with KCNQ2, KCNQ3 and KCNQ4 but not with KCNQ1. This interaction was shown by enhancement of KCNQ2 currents, testing for dominant-negative effects of pore mutants, determining its effects on surface expression and co-immunoprecipitation experiments. Conversely, a KCNQ3-sid(Q1) chimaera no longer affects KCNQ2 but interacts with KCNQ1. We conclude that the si domain suffices to determine the subunit specificity of KCNQ channel assembly.

    EMBO reports 2003;4;1;76-81

  • Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation.

    Yus-Nájera E, Muñoz A, Salvador N, Jensen BS, Rasmussen HB, Defelipe J and Villarroel A

    Instituto Cajal, CSIC, Avenida Dr. Arce 37, 28002 Madrid, Spain.

    The KCNQ family of voltage-dependent non-inactivating K+ channels is composed of five members, four of which (KCNQ2-5) are expressed in the CNS and are responsible for the M-current. Mutations in either KCNQ2 or KCNQ3 lead to a hereditary form of dominant generalized epilepsy. Using specific antisera to the KCNQ2, KCNQ3 and KCNQ5 subunits, we found that KCNQ3 co-immunoprecipitated with KCNQ2 and KCNQ5 subunits, but no association was detected between KCNQ2 and KCNQ5. Intense KCNQ5 immunoreactivity was found to be widely distributed throughout the temporal neocortex and the hippocampal formation. In these structures, both pyramidal and non-pyramidal neurons and a population of glial cells in the white matter expressed the KCNQ5 subunit. In the sclerotic areas of the CA fields of epileptic patients, a marked loss of KCNQ5 immunoreactive pyramidal neurons was found in relation with the loss of neurons in these regions. However, in the regions adjacent to the sclerotic areas, the distribution and intensity of KCNQ5 immunostaining was apparently normal. The widespread distribution of KCNQ5 subunits, its persistence in pharmacoresistant epilepsy, along with the significant role of the M-current in the control of neuronal excitability, makes this protein a possible target for the development of anticonvulsant drugs.

    Neuroscience 2003;120;2;353-64

  • Suggestive evidence for association of two potassium channel genes with different idiopathic generalised epilepsy syndromes.

    Chioza B, Osei-Lah A, Wilkie H, Nashef L, McCormick D, Asherson P and Makoff AJ

    Department of Psychological Medicine, Institute of Psychiatry, Denmark Hill, London, UK.

    Several potassium channel genes have been implicated in epilepsy. We have investigated three such genes, KCNJ3, KCNJ6 and KCNQ2, by association studies using a broad sample of idiopathic generalised epilepsy (IGE) unselected by syndrome. One of the two single nucleotide polymorphisms (SNPs) examined in one of the inward rectifying potassium channel genes, KCNJ3, was associated with IGE by genotype (P=0.0097), while its association by allele was of borderline significance (P=0.051). Analysis of the different clinical subgroups within the IGE sample showed more significant association with the presence of absence seizures (P=0.0041) and which is still significant after correction for multiple testing. Neither SNP in the other rectifying potassium channel gene, KCNJ6, was associated with IGE or any subgroup. None of the three SNPs in the voltage-gated potassium channel gene, KCNQ2, was associated with IGE. However, one SNP was associated with epilepsy with generalised tonic clonic seizures only (P=0.016), as was an SNP approximately 56 kb distant in the closely linked nicotinic acetylcholine gene CHRNA4 (P=0.014). These two SNPs were not in linkage disequilibrium with each other, suggesting that if they are not true associations they have independently occurred by chance. Neither association remains significant after correcting for multiple testing.

    Epilepsy research 2002;52;2;107-16

  • The voltage-gated potassium channel KCNQ2 in Taiwanese children with febrile convulsions.

    Chou IC, Tsai FJ, Huang CC, Lin CC and Tsai CH

    Department of Pediatrics, China Medical College Hospital, Taichung, Taiwan.

    Mutations in the voltage-gated potassium channel genes KCNQ2 and KCNQ3 have been found to cause benign familial neonatal convulsions. Recent studies provided evidence that KCNQ2 and KCNQ3 contribute to the M-current, which regulates the subthreshold electrical excitability in the CNS. Febrile convulsions represent the majority of childhood seizures, and show a strong family history, suggesting a genetic predisposition. By performing an association study, we investigated whether KCNQ2 gene polymorphisms can be used as markers of susceptibility to febrile convulsions. These data suggest that the KCNQ2 gene might not be a useful marker for prediction of the susceptibility of febrile convulsions.

    Neuroreport 2002;13;15;1971-3

  • Calmodulin is an auxiliary subunit of KCNQ2/3 potassium channels.

    Wen H and Levitan IB

    Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

    Calmodulin (CaM) was identified as a KCNQ2 and KCNQ3 potassium channel-binding protein, using a yeast two-hybrid screen. CaM is tethered constitutively to the channel, in the absence or presence of Ca2+, in transfected cells and also coimmunoprecipitates with KCNQ2/3 from mouse brain. The structural elements critical for CaM binding to KCNQ2 lie in two conserved motifs in the proximal half of the channel C-terminal domain. Truncations and point mutations in these two motifs disrupt the interaction. The first CaM-binding motif has a sequence that conforms partially to the consensus IQ motif, but both wild-type CaM and a Ca2+-insensitive CaM mutant bind to KCNQ2. The voltage-dependent activation of the KCNQ2/3 channel also shows no Ca2+ sensitivity, nor is it affected by overexpression of the Ca2+-insensitive CaM mutant. On the other hand, KCNQ2 mutants deficient in CaM binding are unable to generate detectable currents when coexpressed with KCNQ3 in CHO cells, although they are expressed and targeted to the cell membrane and retain the ability to assemble with KCNQ3. A fusion protein containing both of the KCNQ2 CaM-binding motifs competes with the full-length KCNQ2 channel for CaM binding and decreases KCNQ2/3 current density in CHO cells. The correlation of CaM binding with channel function suggests that CaM is an auxiliary subunit of the KCNQ2/3 channel.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;18;7991-8001

  • The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels.

    Yus-Najera E, Santana-Castro I and Villarroel A

    Instituto Cajal, Consejo Superior de Investigaciones, Avenida, Dr. Arce 37, 28002 Madrid, Spain.

    We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two alpha-helices (A and B). These two helices encompass approximately 85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of approximately 130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1-5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.

    The Journal of biological chemistry 2002;277;32;28545-53

  • The DNA sequence and comparative analysis of human chromosome 20.

    Deloukas P, Matthews LH, Ashurst J, Burton J, Gilbert JG, Jones M, Stavrides G, Almeida JP, Babbage AK, Bagguley CL, Bailey J, Barlow KF, Bates KN, Beard LM, Beare DM, Beasley OP, Bird CP, Blakey SE, Bridgeman AM, Brown AJ, Buck D, Burrill W, Butler AP, Carder C, Carter NP, Chapman JC, Clamp M, Clark G, Clark LN, Clark SY, Clee CM, Clegg S, Cobley VE, Collier RE, Connor R, Corby NR, Coulson A, Coville GJ, Deadman R, Dhami P, Dunn M, Ellington AG, Frankland JA, Fraser A, French L, Garner P, Grafham DV, Griffiths C, Griffiths MN, Gwilliam R, Hall RE, Hammond S, Harley JL, Heath PD, Ho S, Holden JL, Howden PJ, Huckle E, Hunt AR, Hunt SE, Jekosch K, Johnson CM, Johnson D, Kay MP, Kimberley AM, King A, Knights A, Laird GK, Lawlor S, Lehvaslaiho MH, Leversha M, Lloyd C, Lloyd DM, Lovell JD, Marsh VL, Martin SL, McConnachie LJ, McLay K, McMurray AA, Milne S, Mistry D, Moore MJ, Mullikin JC, Nickerson T, Oliver K, Parker A, Patel R, Pearce TA, Peck AI, Phillimore BJ, Prathalingam SR, Plumb RW, Ramsay H, Rice CM, Ross MT, Scott CE, Sehra HK, Shownkeen R, Sims S, Skuce CD, Smith ML, Soderlund C, Steward CA, Sulston JE, Swann M, Sycamore N, Taylor R, Tee L, Thomas DW, Thorpe A, Tracey A, Tromans AC, Vaudin M, Wall M, Wallis JM, Whitehead SL, Whittaker P, Willey DL, Williams L, Williams SA, Wilming L, Wray PW, Hubbard T, Durbin RM, Bentley DR, Beck S and Rogers J

    The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK. panos@sanger.ac.uk

    The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

    Nature 2001;414;6866;865-71

  • Cloning and mutation analysis of the human potassium channel KCNQ2 gene promoter.

    Xiao JF, Fischer C, Steinlein OK and JianFeng X

    Institute of Human Genetics, University Hospital Bonn, Wilhelmstr. 31, D-53111 Bonn, Germany.

    Benign familial neonatal convulsions (BFNC) have been previously found to be associated with mutations within the coding region of KCNQ2. We have now cloned and analyzed the promoter region of the human KCNQ2 gene. 5'-RACE identified a transcription start site (TSS) located 200 bp upstream of the ATG start codon. The TSS is located close to a repetitive region containing seven copies of a degenerate 42-mer repeat. Several different luciferase (LUC) reporter plas- mids containing fragments from the KCNQ2 5'-flanking region were constructed and expressed in NT2N and SH-SY5Y cell lines. A core promoter region was found to be located between bp 20 and bp 74 upstream of the TSS. Neither the promoter region nor the repetitive region showed any mutations in 13 index patients from unrelated BFNC families.

    Neuroreport 2001;12;17;3733-9

  • Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K+ channel.

    Dedek K, Kunath B, Kananura C, Reuner U, Jentsch TJ and Steinlein OK

    Zentrum für Molekulare Neurobiologie, Universität Hamburg, D-20246 Hamburg, Germany.

    KCNQ2 and KCNQ3 are two homologous K(+) channel subunits that can combine to form heterotetrameric channels with properties of neuronal M channels. Loss-of-function mutations in either subunit can lead to benign familial neonatal convulsions (BFNC), a generalized, idiopathic epilepsy of the newborn. We now describe a syndrome in which BFNC is followed later in life by myokymia, involuntary contractions of skeletal muscles. All affected members of the myokymia/BFNC family carried a mutation (R207W) that neutralized a charged amino acid in the S4 voltage-sensor segment of KCNQ2. This substitution led to a shift of voltage-dependent activation of KCNQ2 and a dramatic slowing of activation upon depolarization. Myokymia is thought to result from hyperexcitability of the lower motoneuron, and indeed both KCNQ2 and KCNQ3 mRNAs were detected in the anterior horn of the spinal cord where the cells of the lower motoneurons arise. We propose that a difference in firing patterns between motoneurons and central neurons, combined with the drastically slowed voltage activation of the R207W mutant, explains why this particular KCNQ2 mutant causes myokymia in addition to BFNC.

    Proceedings of the National Academy of Sciences of the United States of America 2001;98;21;12272-7

  • Differential expression of kcnq2 splice variants: implications to m current function during neuronal development.

    Smith JS, Iannotti CA, Dargis P, Christian EP and Aiyar J

    Departments of Neuroscience and Enabling Science and Technology, AstraZeneca Pharmaceuticals, Wilmington, Delaware 19803, USA. jeff.smith@astrazeneca.com

    The KCNQ family of K(+) channels has been implicated in several cardiac and neurological disease pathologies. KCNQ2 (Q2) is a brain-derived gene, which in association with KCNQ3 (Q3) has been shown to provide a molecular basis for the neuronal M current. We have cloned a long (Q2L) and a short (Q2S) splice variant of the human KCNQ2 gene; these variants differ in their C-terminal tail. Northern blot analysis reveals that Q2L is preferentially expressed in differentiated neurons, whereas the Q2S transcript is prominent in fetal brain, undifferentiated neuroblastoma cells, and brain tumors. Q2L, transfected into mammalian cells, produces a slowly activating, noninactivating voltage-gated K(+) current that is blocked potently by tetraethylammonium (TEA; IC(50), 0.14 mm). Q2S on the other hand produces no measurable potassium currents. Cotransfection of Q2S with either Q2L, Q3, or Q2L/Q3 heteromultimers results in attenuation of K(+) current, the suppression being most profound for Q3. Inclusion of Q2S in the heteromultimer also positively shifts the voltage dependence of current activation and alters affinity for the TEA block, suggesting that under these conditions, some Q2S subunits incorporate into functional channels on the plasma membrane. In view of the crucial role of M currents in modulating neuronal excitability, our findings provide important insight into the functional consequences of differential expression of KCNQ2 splice variants: dampened potassium conductances in the developing brain could shape firing repertoires to provide cues for proliferation rather than differentiation.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;4;1096-103

  • Benign familial neonatal convulsions (BFNC) resulting from mutation of the KCNQ2 voltage sensor.

    Miraglia del Giudice E, Coppola G, Scuccimarra G, Cirillo G, Bellini G and Pascotto A

    Department of Pediatrics, Second University of Naples, Italy.

    Benign familial neonatal convulsions (BFNC) is a rare autosomal inherited epilepsy. We studied the KCNQ2 coding region in a large, four-generation, Italian family with BFNC. A missense mutation C686T predicting the change of one of the innermost arginine (R214W) of the key functional voltage sensor (S4 helix), has been found in all affected members. This substitution probably reduces the movement of the voltage sensor that precedes channel opening during voltage-dependent activation. Several mutations affecting the trans-membrane domain and the pore region of the K+ channels belonging to the KQT-like family have been described in some human diseases associated with altered regulation of cellular excitability (ie BFNC, some LQT syndromes and DFNA2). R214W represents the first mutation involving the region of the voltage sensor.

    European journal of human genetics : EJHG 2000;8;12;994-7

  • M-type KCNQ2-KCNQ3 potassium channels are modulated by the KCNE2 subunit.

    Tinel N, Diochot S, Lauritzen I, Barhanin J, Lazdunski M and Borsotto M

    Institut de Pharmacologie Moléculaire et Cellulaire, CNRS-UPR 411, Valbonne, France.

    KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K+ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2-KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.

    FEBS letters 2000;480;2-3;137-41

  • Retigabine, a novel anti-convulsant, enhances activation of KCNQ2/Q3 potassium channels.

    Wickenden AD, Yu W, Zou A, Jegla T and Wagoner PK

    ICAgen Inc., Durham, North Carolina 27702, USA. awickenden@icagen.com

    Retigabine [N-(2-amino-4-[fluorobenzylamino]-phenyl) carbamic acid; D-23129] is a novel anticonvulsant, unrelated to currently available antiepileptic agents, with activity in a broad range of seizure models. In the present study, we sought to determine whether retigabine could enhance current through M-like currents in PC12 cells and KCNQ2/Q3 K(+) channels expressed in Chinese hamster ovary cells (CHO-KCNQ2/Q3). In differentiated PC12 cells, retigabine enhanced a linopirdine-sensitive current. The effect of retigabine was associated with a slowing of M-like tail current deactivation in these cells. Retigabine (0.1 to 10 microM) induced a potassium current and hyperpolarized CHO cells expressing KCNQ2/Q3 cells but not in wild-type cells. Retigabine-induced currents in CHO-KCNQ2/Q3 cells were inhibited by 60.6 +/- 11% (n = 4) by the KCNQ2/Q3 blocker, linopirdine (10 microM), and 82.7 +/- 5.4% (n = 4) by BaCl(2) (10 mM). The mechanism by which retigabine enhanced KCNQ2/Q3 currents involved large, drug-induced, leftward shifts in the voltage dependence of channel activation (-33.1 +/- 2.6 mV, n = 4, by 10 microM retigabine). Retigabine shifted the voltage dependence of channel activation with an EC(50) value of 1.6 +/- 0.3 microM (slope factor was 1.2 +/- 0.1, n = 4 to 5 cells per concentration). Retigabine (0.1 to 10 microM) also slowed the rate of channel deactivation, predominantly by increasing the contribution of a slowly deactivating tail current component. Our findings identify KCNQ2/Q3 channels as a molecular target for retigabine and suggest that activation of KCNQ2/Q3 channels may be responsible for at least some of the anticonvulsant activity of this agent.

    Molecular pharmacology 2000;58;3;591-600

  • Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine.

    Main MJ, Cryan JE, Dupere JR, Cox B, Clare JJ and Burbidge SA

    Molecular Pharmacology, Neuroscience, Glaxo-Wellcome Research & Development, Stevenage, Hertfordshire, United Kingdom. mjm37276@glaxowellcome.co.uk

    Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination in Xenopus oocytes. Application of 10 microM retigabine to oocytes expressing the KCNQ2/3 heteromeric channel shifted both the activation threshold and voltage for half-activation by approximately 20 mV in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between -80 mV and +20 mV. Retigabine also had a marked effect on KCNQ current kinetics, increasing the rate of channel activation but slowing deactivation at a given test potential. Similar effects of retigabine were observed in oocytes expressing KCNQ2 alone, suggesting that KCNQ2 may be the molecular target of retigabine. Membrane potential recordings in oocytes expressing the KCNQ2/3 heteromeric channel showed that application of retigabine leads to a concentration-dependent hyperpolarization of the oocyte, from a resting potential of -63 mV under control conditions to -85 mV in the presence of 100 microM retigabine (IC(50) = 5.2 microM). In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.

    Molecular pharmacology 2000;58;2;253-62

  • Surface expression and single channel properties of KCNQ2/KCNQ3, M-type K+ channels involved in epilepsy.

    Schwake M, Pusch M, Kharkovets T and Jentsch TJ

    Zentrum für Molekulare Neurobiologie Hamburg (ZMNH), Universität Hamburg, Martinistrasse 85, D-20246 Hamburg, Germany.

    Mutations in either KCNQ2 or KCNQ3 underlie benign familial neonatal convulsions (BFNC), an inherited epilepsy. The corresponding proteins are co-expressed in broad regions of the brain and associate to heteromeric K(+) channels. These channels mediate M-type currents that regulate neuronal excitability. We investigated the basis for the increase in currents seen after co-expressing these subunits in Xenopus oocytes. Noise analysis and single channel recordings revealed a conductance of approximately 18 pS for KCNQ2 and approximately 7 pS for KCNQ3. Different conductance levels (ranging from 8 to 22 pS) were seen upon co-expression. Their weighted average is close to that obtained by noise analysis (16 pS). The open probability of heteromeric channels was not increased significantly. Co-expression of both subunits increased the surface expression of KCNQ2 and KCNQ3 by factors of 5 and >10, respectively. A KCNQ2 mutant associated with BFNC that has a truncated cytoplasmic carboxyl terminus did not reach the surface and failed to stimulate KCNQ3 surface expression. By contrast, several BFNC-associated missense mutations in KCNQ2 or KCNQ3 did not alter their surface expression. Thus, the increase in currents seen upon co-expressing KCNQ2 and KCNQ3 is predominantly due to an increase in surface expression, which is dependent on an intact carboxyl terminus.

    Funded by: Telethon: 1079

    The Journal of biological chemistry 2000;275;18;13343-8

  • Colocalization and coassembly of two human brain M-type potassium channel subunits that are mutated in epilepsy.

    Cooper EC, Aldape KD, Abosch A, Barbaro NM, Berger MS, Peacock WS, Jan YN and Jan LY

    Department of Neurology, Howard Hughes Medical Institute, University of California, San Francisco, CA 94143, USA.

    Acetylcholine excites many central and autonomic neurons through inhibition of M-channels, slowly activating, noninactivating voltage-gated potassium channels. We here provide information regarding the in vivo distribution and biochemical characteristics of human brain KCNQ2 and KCNQ3, two channel subunits that form M-channels when expressed in vitro, and, when mutated, cause the dominantly inherited epileptic syndrome, benign neonatal familial convulsions. KCNQ2 and KCNQ3 proteins are colocalized in a somatodendritic pattern on pyramidal and polymorphic neurons in the human cortex and hippocampus. Immunoreactivity for KCNQ2, but not KCNQ3, is also prominent in some terminal fields, suggesting a presynaptic role for a distinct subgroup of M-channels in the regulation of action potential propagation and neurotransmitter release. KCNQ2 and KCNQ3 can be coimmunoprecipitated from brain lysates. Further, KCNQ2 and KCNQ3 are coassociated with tubulin and protein kinase A within a Triton X-100-insoluble protein complex. This complex is not associated with low-density membrane rafts or with N-methyl-d-aspartate receptors, PSD-95 scaffolding proteins, or other potassium channels tested. Our studies thus provide a view of a signaling complex that may be important for cognitive function as well as epilepsy. Analysis of this complex may shed light on the unknown transduction pathway linking muscarinic acetylcholine receptor activation to M-channel inhibition.

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;9;4914-9

  • The novel anticonvulsant retigabine activates M-currents in Chinese hamster ovary-cells tranfected with human KCNQ2/3 subunits.

    Rundfeldt C and Netzer R

    Department of Pharmacology, Arzneimittelwerk Dresden GmbH, Corporate R&D, ASTA Medica Group, Meibetaner Strasse 35, D-01445, Radebeul, Germany. dr_chris.rundfeldt@astamedica.de

    Retigabine (D-23129) is a novel antiepileptic compound with broad spectrum and potent anticonvulsant properties, both in vitro and in vivo. The compound was shown to activate a K(+) current in neuronal cells. The pharmacology of the induced current displays concordance with the published pharmacology of the M-channel, which recently was correlated to the KCNQ2/3 K(+) channel heteromultimere. We examined the effect of retigabine on KCNQ2/3 expressed in Chinese hamster ovary cells. The compound concentration-dependently activated a K(+) current in transfected cells clamped at -50 mV. The activation was induced by a shift of the opening threshold to more negative potentials. The effect was not mediated by an interaction with the cAMP modulatory site and could be partially blocked by the M-channel antagonist linopirdine. The data display that retigabine is the first described M-channel agonist and support the hypothesis that M-channel agonism is a new mode of action for anticonvulsant drugs. Since the function of this channel is reduced in a hereditary epilepsy syndrome, retigabine may be the first anticonvulsant to directly target the deficit observed in a channelopathy.

    Neuroscience letters 2000;282;1-2;73-6

  • Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K(+) channels that underlie the neuronal M current.

    Shapiro MS, Roche JP, Kaftan EJ, Cruzblanca H, Mackie K and Hille B

    Department of Physiology, University of Washington School of Medicine, Seattle, Washington 98195, USA. mshapiro@u.washington.edu

    Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K(+) current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M(1) muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca(2+) transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca(2+) signals were abolished by the combined use of strong intracellular Ca(2+) buffers, an inhibitor of IP(3) receptors, and thapsigargin to deplete Ca(2+) stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca(2+) as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.

    Funded by: NIAMS NIH HHS: AR17803; NIDA NIH HHS: DA00286; NINDS NIH HHS: NS081734; ...

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;5;1710-21

  • Inhibition of KCNQ1-4 potassium channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors.

    Selyanko AA, Hadley JK, Wood IC, Abogadie FC, Jentsch TJ and Brown DA

    Department of Pharmacology, University College London, UK. a.selyanko@ucl.ac.uk

    1. KCNQ1-4 potassium channels were expressed in mammalian Chinese hamster ovary (CHO) cells stably transfected with M1 muscarinic acetylcholine receptors and currents were recorded using the whole-cell perforated patch technique and cell-attached patch recording. 2. Stimulation of M1 receptors by 10 microM oxotremorine-M (Oxo-M) strongly reduced (to 0-10%) currents produced by KCNQ1-4 subunits expressed individually and also those produced by KCNQ2 + KCNQ3 and KCNQ1 + KCNE1 heteromers, which are thought to generate neuronal M-currents (IK,M) and cardiac slow delayed rectifier currents (IK,s), respectively. 3. The activity of KCNQ2 + KCNQ3, KCNQ2 and KCNQ3 channels recorded with cell-attached pipettes was strongly and reversibly reduced by Oxo-M applied to the extra-patch membrane. 4. It is concluded that M1 receptors couple to all known KCNQ subunits and that inhibition of KCNQ2 + KCNQ3 channels, like that of native M-channels, requires a diffusible second messenger.

    The Journal of physiology 2000;522 Pt 3;349-55

  • Two types of K(+) channel subunit, Erg1 and KCNQ2/3, contribute to the M-like current in a mammalian neuronal cell.

    Selyanko AA, Hadley JK, Wood IC, Abogadie FC, Delmas P, Buckley NJ, London B and Brown DA

    Department of Pharmacology, University College London, London, WC1E 6BT, United Kingdom.

    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.

    Funded by: Wellcome Trust

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1999;19;18;7742-56

  • Structural and mutational analysis of KCNQ2, the major gene locus for benign familial neonatal convulsions.

    Biervert C and Steinlein OK

    Institute for Human Genetics, University of Bonn, Germany.

    Mutations in the voltage-gated potassium channel gene KCNQ2 on chromosome 20q13.3 are responsible for benign familial neonatal convulsions (BFNC), a rare monogenic idiopathic epilepsy. Here we report the determination of the detailed genomic structure of KCNQ2, and use of this information in mutational analysis. There are at least 18 exons, occupying more than 50 kb of genomic DNA. Several formerly unknown polymorphisms and splice variants as well as a new single base pair deletion mutation of unusual localization are described. In addition to facilitating more effective mutation detection among BFNC patients, the results presented here provide the basis for analysing the role of KCNQ2 in other types of epilepsy.

    Human genetics 1999;104;3;234-40

  • Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy.

    Schroeder BC, Kubisch C, Stein V and Jentsch TJ

    Zentrum für Molekulare Neurobiologie Hamburg, Universität Hamburg, Germany.

    Epilepsy affects more than 0.5% of the world's population and has a large genetic component. It is due to an electrical hyperexcitability in the central nervous system. Potassium channels are important regulators of electrical signalling, and benign familial neonatal convulsions (BFNC), an autosomal dominant epilepsy of infancy, is caused by mutations in the KCNQ2 or the KCNQ3 potassium channel genes. Here we show that KCNQ2 and KCNQ3 are distributed broadly in brain with expression patterns that largely overlap. Expression in Xenopus oocytes indicates the formation of heteromeric KCNQ2/KCNQ3 potassium channels with currents that are at least tenfold larger than those of the respective homomeric channels. KCNQ2/KCNQ3 currents can be increased by intracellular cyclic AMP, an effect that depends on an intact phosphorylation site in the KCNQ2 amino terminus. KCNQ2 and KCNQ3 mutations identified in BFNC pedigrees compromised the function of the respective subunits, but exerted no dominant-negative effect on KCNQ2/KCNQ3 heteromeric channels. We predict that a 25% loss of heteromeric KCNQ2/KCNQ3-channel function is sufficient to cause the electrical hyperexcitability in BFNC. Drugs raising intracellular cAMP may prove beneficial in this form of epilepsy.

    Nature 1998;396;6712;687-90

  • KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel.

    Wang HS, Pan Z, Shi W, Brown BS, Wymore RS, Cohen IS, Dixon JE and McKinnon D

    Institute of Molecular Cardiology, Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook, NY 11794, USA.

    The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current.

    Science (New York, N.Y.) 1998;282;5395;1890-3

  • The KCNQ2 potassium channel: splice variants, functional and developmental expression. Brain localization and comparison with KCNQ3.

    Tinel N, Lauritzen I, Chouabe C, Lazdunski M and Borsotto M

    Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, Valbonne, France.

    Benign familial neonatal convulsions, an autosomal dominant epilepsy of newborns, are linked to mutations affecting two six-transmembrane potassium channels, KCNQ2 and KCNQ3. We isolated four splice variants of KCNQ2 in human brain. Two forms generate, after transient expression in COS cells, a potassium-selective current similar to the KCNQ1 current. L-735,821, a benzodiazepine molecule which inhibits the KCNQ1 channel activity (EC50 = 0.08 microM), also blocks KCNQ2 currents (EC50 = 1.5 microM). Using in situ hybridization, KCNQ2 and KCNQ3 have been localized within the central nervous system, in which they are expressed in the same areas, mainly in the hippocampus, the neocortex and the cerebellar cortex. During brain development, KCNQ3 is expressed later than KCNQ2.

    FEBS letters 1998;438;3;171-6

  • Functional expression of two KvLQT1-related potassium channels responsible for an inherited idiopathic epilepsy.

    Yang WP, Levesque PC, Little WA, Conder ML, Ramakrishnan P, Neubauer MG and Blanar MA

    Department of Cardiovascular Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.

    Benign familial neonatal convulsions (BFNC), a class of idiopathic generalized epilepsy, is an autosomal dominantly inherited disorder of newborns. BFNC has been linked to mutations in two putative K+ channel genes, KCNQ2 and KCNQ3. Amino acid sequence comparison reveals that both genes share strong homology to KvLQT1, the potassium channel encoded by KCNQ1, which is responsible for over 50% of inherited long QT syndrome. Here we describe the cloning, functional expression, and characterization of K+ channels encoded by KCNQ2 and KCNQ3 cDNAs. Individually, expression of KCNQ2 or KCNQ3 in Xenopus oocytes elicits voltage-gated, rapidly activating K+-selective currents similar to KCNQ1. However, unlike KCNQ1, KCNQ2 and KCNQ3 currents are not augmented by coexpression with the KCNQ1 beta subunit, KCNE1 (minK, IsK). Northern blot analyses reveal that KCNQ2 and KCNQ3 exhibit similar expression patterns in different regions within the brain. Interestingly, coexpression of KCNQ2 and KCNQ3 results in a substantial synergistic increase in current amplitude. Coexpression of KCNE1 with the two channels strongly suppressed current amplitude and slowed kinetics of activation. The pharmacological and biophysical properties of the K+ currents observed in the coinjected oocytes differ somewhat from those observed after injecting either KCNQ2 or KCNQ3 by itself. The functional interaction between KCNQ2 and KCNQ3 provides a framework for understanding how mutations in either channel can cause a form of idiopathic generalized epilepsy.

    The Journal of biological chemistry 1998;273;31;19419-23

  • A potassium channel mutation in neonatal human epilepsy.

    Biervert C, Schroeder BC, Kubisch C, Berkovic SF, Propping P, Jentsch TJ and Steinlein OK

    Institute for Human Genetics, University of Bonn, Bonn, Germany.

    Benign familial neonatal convulsions (BFNC) is an autosomal dominant epilepsy of infancy, with loci mapped to human chromosomes 20q13.3 and 8q24. By positional cloning, a potassium channel gene (KCNQ2) located on 20q13.3 was isolated and found to be expressed in brain. Expression of KCNQ2 in frog (Xenopus laevis) oocytes led to potassium-selective currents that activated slowly with depolarization. In a large pedigree with BFNC, a five-base pair insertion would delete more than 300 amino acids from the KCNQ2 carboxyl terminus. Expression of the mutant channel did not yield measurable currents. Thus, impairment of potassium-dependent repolarization is likely to cause this age-specific epileptic syndrome.

    Science (New York, N.Y.) 1998;279;5349;403-6

  • A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns.

    Singh NA, Charlier C, Stauffer D, DuPont BR, Leach RJ, Melis R, Ronen GM, Bjerre I, Quattlebaum T, Murphy JV, McHarg ML, Gagnon D, Rosales TO, Peiffer A, Anderson VE and Leppert M

    Department of Human Genetics, University of Utah, Salt Lake City 84112, USA.

    Idiopathic generalized epilepsies account for about 40% of epilepsy up to age 40 and commonly have a genetic basis. One type is benign familial neonatal convulsions (BFNC), a dominantly inherited disorder of newborns. We have identified a sub-microscopic deletion of chromosome 20q13.3 that co-segregates with seizures in a BFNC family. Characterization of cDNAs spanning the deleted region identified one encoding a novel voltage-gated potassium channel, KCNQ2, which belongs to a new KQT-like class of potassium channels. Five other BFNC probands were shown to have KCNQ2 mutations, including two transmembrane missense mutations, two frameshifts and one splice-site mutation. This finding in BFNC provides additional evidence that defects in potassium channels are involved in the mammalian epilepsy phenotype.

    Funded by: NCI NIH HHS: 5P30 CA42014; NCRR NIH HHS: M01 RR00064; NINDS NIH HHS: R01 NS32666

    Nature genetics 1998;18;1;25-9

  • Identification and cloning of neuroblastoma-specific and nerve tissue-specific genes through compiled expression profiles.

    Yokoyama M, Nishi Y, Yoshii J, Okubo K and Matsubara K

    Life Science Research Laboratory, Japan Tobacco, Inc., Kanagawa, Japan.

    An expression profile of active genes in a human neuroblastoma cell line CHP134 was obtained by collecting 1222 partial sequences from a 3'-directed cDNA library representing a non-biased mRNA population. By comparing this expression profile with the compiled profiles of multiple tissues, several novel gene transcripts that appeared only in the profile of the neuroblastoma cell line were identified. Further analyses by Northern blotting revealed two specific cDNA clones that are expressed in most of the human neuroblastomas examined, and three that are in some of the human neuroblastoma cell lines as well as in the adult human brain. Full-size cDNAs were cloned using these five partial cDNA sequences as probes and sequenced. A database search revealed that they are all novel and unique sequences: one 1706 sharing some amino acid sequence similarities with a cytoskeletal protein, two clones likely to be transcriptional factors, a clone that has characteristic potassium channel properties, and a clone that is non-homologous to any one of the known proteins. Thus, we argue that the collection of 3'-directed cDNA sequences in combination with the compiled expression profiles of active genes in multiple tissues is a powerful tool for discovering novel genes that are specifically expressed in a given cell or tissue, in this case neuroblastomas and/or nerve tissue.

    DNA research : an international journal for rapid publication of reports on genes and genomes 1996;3;5;311-20

Gene lists (5)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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