[Research progress on M-type potassium channel].
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Animals↗
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Results for “KCNQ2 Potassium Channel”
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KCNQ2 and KCNQ3 potassium channel genes in benign familial neonatal convulsions: expansion of the functional and mutation spectrum.
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.
Animals↗
(S)-N-[1-(3-morpholin-4-ylphenyl)ethyl]- 3-phenylacrylamide: an orally bioavailable KCNQ2 opener with significant activity in a cortical spreading depression model of migraine.
(S)-N-[1-(3-Morpholin-4-ylphenyl)ethyl]-3-phenylacrylamide (2) was synthesized as an orally bioavailable KCNQ2 potassium channel opener. In a rat model of migraine, 2 demonstrated significant oral activity in reducing the total number of cortical spreading depressions induced by potassium chloride.
Acrylamides↗
Stoichiometry of expressed KCNQ2/KCNQ3 potassium channels and subunit composition of native ganglionic M channels deduced from block by tetraethylammonium.
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.
Animals↗
The identification and characterization of a noncontinuous calmodulin-binding site in noninactivating voltage-dependent KCNQ potassium channels.
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.
Adult↗
Identification of specific pore residues mediating KCNQ1 inactivation. A novel mechanism for long QT syndrome.
KCNQ1 inactivation bears electrophysiological characteristics different from classical N- and C-type inactivation in Shaker-like potassium channels. However, the molecular site of KCNQ1 inactivation has not yet been determined. KCNQ2 channels do not exert a fast inactivation in contrast to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and KCNQ2 in Xenopus oocytes, we mapped the region of this inactivation to transmembrane domain S5 and the pore loop H5 and finally narrowed down the site to positions Gly(272) and Val(307) in KCNQ1. Exchanging these two amino acids individually with the analogous KCNQ2 residue abolished inactivation. Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by mutagenesis in the corresponding region, confirming its relevance for the inactivation process. As KCNQ1 inactivation involves the regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac I(Ks) channels comprising KCNQ1 and accessory MinK subunits do not inactivate because of the functional interaction of KCNQ1 with MinK. Mutations in KCNQ1 can lead to long QT1 syndrome, an inherited form of arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation. Here we show that when expressing mutant I(Ks) channels formed from KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation. This results in smaller repolarizing currents in the heart and therefore represents a novel mechanism leading to long QT syndrome.
Amino Acid Sequence↗
A KCNQ2 splice site mutation causing benign neonatal convulsions in a Scottish family.
Benign familial neonatal convulsions (BFNC) are one of the rare idiopathic epilepsies with autosomal dominant mode of inheritance. Two voltage-gated potassium channels, KCNQ2 on chromosome 20q13.3 and KCNQ3 on 8q24, have been recently identified as the genes responsible for BFNC. Here we describe a large family with BFNC in which we found a previously undescribed mutation in the KCNQ2 gene. A 1187(+2)T/G nucleotide exchange affects the conserved donor splice site motif in intron 9. This mutation can be predicted to give rise to aberrant splicing of the primary transcript. There was a wide range of clinical manifestations in this family. An unusual clinical feature is the occurrence of partial seizures in later life with corresponding focal neurological deficits.
Chromosome Aberrations↗
Inhibition of KCNQ1-4 potassium channels expressed in mammalian cells via M1 muscarinic acetylcholine receptors.
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.
Animals↗
Molecular correlates of the M-current in cultured rat hippocampal neurons.
M-type K(+) currents (I(K(M))) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K(+) channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I(K(M)) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I(K(M)) with an IC(50) of 3.6 +/- 1.5 microM. In 70 % of these cells, TEA fully suppressed I(K(M)) with an IC(50) of 0.7 +/- 0.1 mM. In the remaining cells, TEA maximally reduced I(K(M)) by only 59.7 +/- 5.2 % with an IC(50) of 1.4 +/- 0.3 mM; residual I(K(M)) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I(K(M)) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.
Animals↗
Activation of expressed KCNQ potassium currents and native neuronal M-type potassium currents by the anti-convulsant drug retigabine.
Retigabine [D-23129; N-(2-amino-4-(4-fluorobenzylamino)-phenyl) carbamic acid ethyl ester] is a novel anticonvulsant compound that is now in clinical phase II development. It has previously been shown to enhance currents generated by KCNQ2/3 K(+) channels when expressed in Chinese hamster ovary (CHO) cells (Main et al., 2000; Wickenden et al., 2000). In the present study, we have compared the actions of retigabine on KCNQ2/3 currents with those on currents generated by other members of the KCNQ family (homomeric KCNQ1, KCNQ2, KCNQ3, and KCNQ4 channels) expressed in CHO cells and on the native M current in rat sympathetic neurons [thought to be generated by KCNQ2/3 channels (Wang et al., 1998)]. Retigabine produced a hyperpolarizing shift of the activation curves for KCNQ2/3, KCNQ2, KCNQ3, and KCNQ4 currents with differential potencies in the following order: KCNQ3 > KCNQ2/3 > KCNQ2 > KCNQ4, as measured either by the maximum hyperpolarizing shift in the activation curves or by the EC(50) values. In contrast, retigabine did not enhance cardiac KCNQ1 currents. Retigabine also produced a hyperpolarizing shift in the activation curve for native M channels in rat sympathetic neurons. The retigabine-induced current was inhibited by muscarinic receptor stimulation, with similar agonist potency but 25% reduced maximum effect. In unclamped neurons, retigabine produced a hyperpolarization and reduced the number of action potentials produced by depolarizing current injections, without change in action potential configuration.
Action Potentials↗
A potassium channel mutation in neonatal human epilepsy.
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.
Action Potentials↗
Localization of KCNQ5 in the normal and epileptic human temporal neocortex and hippocampal formation.
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.
Adolescent↗
KCNQ/M-currents contribute to the resting membrane potential in rat visceral sensory neurons.
The M-current is a slowly activating, non-inactivating potassium current that has been shown to be present in numerous cell types. In this study, KCNQ2, Q3 and Q5, the molecular correlates of M-current in neurons, were identified in the visceral sensory neurons of the nodose ganglia from rats through immunocytochemical studies. All neurons showed expression of each of the three proteins. In voltage clamp studies, the cognition-enhancing drug linopirdine (1-50 microM) and its analogue, XE991 (10 microM), quickly and irreversibly blocked a small, slowly activating current that had kinetic properties similar to KCNQ/M-currents. This current activated between -60 and -55 mV, had a voltage-dependent activation time constant of 208 +/- 12 ms at -20 mV, a deactivation time constant of 165 +/- 24 ms at -50 mV and V1/2 of -24 +/- 2 mV, values which are consistent with previous reports for endogenous M-currents. In current clamp studies, these drugs also led to a depolarization of the resting membrane potential at values as negative as -60 mV. Flupirtine (10-20 microM), an M-current activator, caused a 3-14 mV leftward shift in the current-voltage relationship and also led to a hyperpolarization of resting membrane potential. These data indicate that the M-current is present in nodose neurons, is activated at resting membrane potential and that it is physiologically important in regulating excitability by maintaining cells at negative voltages.
Aminopyridines↗
KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel.
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.
Adult↗
Potassium channels: how genetic studies of epileptic syndromes open paths to new therapeutic targets and drugs.
How can epilepsy gene hunting lead to better care for patients with epilepsy? Lessons may be learned from the progress made by identifying the mutated genes that cause Benign Familial Neonatal Convulsions (BFNC). In 1998, a decade of clinical and laboratory-based genetics work resulted in the cloning of the KCNQ2 potassium channel gene at the BFNC locus on chromosome 20. Subsequently, computer "mining" of public DNA databases allowed the rapid identification of three more brain KCNQ genes. Mutations in each of these additional genes were implicated as causes of human hereditary diseases: epilepsy (KCNQ3), deafness (KCNQ4), and, possibly, retinal degeneration (KCNQ5). Physiologists discovered that the KCNQ genes encoded subunits of the "M-channel," a type of potassium channel known to control repetitive neuronal discharges. Finally, pharmacologists discovered that retigabine, a novel anticonvulsant with a broad but distinctive efficacy profile in animal studies, was a potent KCNQ channel opener. These studies suggest that KCNQ channels may be an important new class of targets for anticonvulsant therapies. The efficacy of retigabine is currently being tested in multicenter clinical trials; identification of its molecular targets will allow it to be more efficiently exploited as a "lead compound." Cloned human KCNQ channels can now be expressed in cultured cells for "high-throughput" screening of drug candidates. Ongoing studies of the KCNQ channels in humans and animal models will refine our understanding of how M-channels control excitability at the cellular, network, and behavioral levels, and may reveal additional targets for therapeutic manipulation.
Animals↗
Properties of single M-type KCNQ2/KCNQ3 potassium channels expressed in mammalian cells.
1. The single channel properties of KCNQ2/KCNQ3 channels underlying neuronal voltage-dependent M-type potassium currents were studied in cell-attached patches from transfected Chinese hamster ovary (CHO) cells. Macroscopic currents produced by homo- and heteromeric KCNQ2/KCNQ3 channels were measured using the perforated-patch whole-cell technique. 2. Compared with heteromeric KCNQ2 + KCNQ3 channels, homomeric KCNQ2 channels had lower slope conductance (9.0 +/- 0.3 and 5.8 +/- 0.3 pS, respectively) and open probability at 0 mV (0.30 +/- 0.07 and 0.15 +/- 0.03, respectively), consistent with their 3.8-fold smaller macroscopic currents. By contrast, homomeric KCNQ3 channels had the same slope conductance (9.0 +/- 1.1 pS) as KCNQ2 + KCNQ3 channels, and higher open probability (0.59 +/- 0.11), inconsistent with their 12.7-fold smaller macroscopic currents. Thus, KCNQ2 and KCNQ3 subunits may play different roles in the expression of M-type currents, with KCNQ2 ensuring surface expression of underlying channels and KCNQ3 modifying their function. 3. Both in homo- and heteromeric KCNQ2/KCNQ3 channels the shut time distributions were fitted with three, and the open time distributions with two, exponential components. By measuring these and other parameters (e.g. conductance and open probability) KCNQ2/ KCNQ3 channels can be shown to resemble previously characterised neuronal M-type channels.
Animals↗
Neonatal seizures with tonic clonic sequences and poor developmental outcome.
Seizures consisting of a tonic followed by a clonic phase have rarely been described in neonates and are not included in the current classifications of neonatal seizures. Our video archive of 105 neonates with seizures or suspected seizures revealed six neonates with such tonic clonic or tonic myoclonic sequences. Two of those neonates had pyridoxine dependent seizures. The other four neonates had drug refractory seizures and demonstrated similarities in electro-clinical pattern, clinical course and outcome. Their seizures started with tonic posturing and after 10-20s tonic posturing was superimposed by focal or multifocal cloni or myocloni. Ictal EEG started with voltage attenuation followed by bilateral or alternating focal epileptic discharges. The interictal EEG was abnormal. One child died, while the other three children became seizure free but had severe motor delay and mental retardation. In one of those three children, a de novo missense mutation was detected in the voltage gated potassium channel gene KCNQ2, indicating a genetic relationship between drug refractory neonatal seizures of unknown etiology with tonic clonic or myoclonic sequences and the well-known syndrome of benign familial neonatal convulsions (BFNC).
Child↗
Genes and mutations in idiopathic epilepsy.
Partial or generalized idiopathic epilepsies, which account for up to 40% of all epilepsies, are characterized by a mostly benign course and no apparent etiology other than a genetic predisposition. So far, the genetic defects underlying three different idiopathic epilepsy syndromes have been identified: mutations in the CHRNA4- or CHRNB subunits of the neuronal nicotinic acetylcholine receptor are found in familial nocturnal frontal lobe epilepsy, while defects in the voltage-gated potassium channels KCNQ2 and KCNQ3 have recently been identified in benign familial neonatal convulsions. The syndrome of "generalized epilepsy with febrile seizures plus" can be caused by mutations affecting the voltage-gated sodium channel subunits SCN1B and SCN1A or the gamma 2-subunit of the GABA(A) receptor. The results of recent molecular studies contributed largely to our understanding of the etiology and pathophysiology of idiopathic epilepsies.
Epilepsies, Partial↗