Mutations In Kv7 Research Articles

Novel loss-of-function mutations in KV7.2, and their possible involvement in epilepsy

Novel loss-of-function mutations in KV7.2, and their possible involvement in epilepsy

A benzodiazepine activator locks Kv7.1 channels open by electro-mechanical uncoupling

Loss-of-function mutations in Kv7.1 often lead to long QT syndrome (LQTS), a cardiac repolarization disorder associated with arrhythmia and subsequent sudden cardiac death. The discovery of agonistic IKs modulators may offer a new potential strategy in pharmacological treatment of this disorder. The benzodiazepine derivative (R)-L3 potently activates Kv7.1 channels and shortens action potential duration, thus may represent a starting point for drug development. However, the molecular mechanisms underlying modulation by (R)-L3 are still unknown. By combining alanine scanning mutagenesis, non-canonical amino acid incorporation, voltage-clamp electrophysiology and fluorometry, and in silico protein modelling, we show that (R)-L3 not only stimulates currents by allosteric modulation of the pore domain but also alters the kinetics independently from the pore domain effects. We identify novel (R)-L3-interacting key residues in the lower S4-segment of Kv7.1 and observed an uncoupling of the outer S4 segment with the inner S5, S6 and selectivity filter segments.

Activation of SGK1.1 Upregulates the M-current in the Presence of Epilepsy Mutations.

In the central nervous system, the M-current plays a critical role in regulating subthreshold electrical excitability of neurons, determining their firing properties and responsiveness to synaptic input. The M-channel is mainly formed by subunits Kv7.2 and Kv7.3 that co-assemble to form a heterotetrametric channel. Mutations in Kv7.2 and Kv7.3 are associated with hyperexcitability phenotypes including benign familial neonatal epilepsy (BFNE) and neonatal epileptic encephalopathy (NEE). SGK1.1, the neuronal isoform of the serum and glucocorticoids-regulated kinase 1 (SGK1), increases M-current density in neurons, leading to reduced excitability and protection against seizures. Herein, using two-electrode voltage clamp on Xenopus laevis oocytes, we demonstrate that SGK1.1 selectively activates heteromeric Kv7 subunit combinations underlying the M-current. Importantly, activated SGK1.1 increases M-channel activity in the presence of two different epilepsy mutations found in Kv7.2, R207W and A306T. In addition, proximity ligation assays in the N2a cell line allowed us to address the effect of these mutations on Kv7-SGK1.1-Nedd4 molecular associations, a proposed pathway underlying augmentation of M-channel activity by SGK1.1

The S2–S3 Loop of Kv7.4 Channels Is Essential for Calmodulin Regulation of Channel Activation

Kv7.4 (KCNQ4) voltage-gated potassium channels control excitability in the inner ear and the central auditory pathway. Mutations in Kv7.4 channels result in inherited progressive deafness in humans. Calmodulin (CaM) is crucial for regulating Kv7 channels, but how CaM affects Kv7 activity has remained unclear. Here, based on electrophysiological recordings, we report that the third EF hand (EF3) of CaM controls the calcium-dependent regulation of Kv7.4 activation and that the S2–S3 loop of Kv7.4 is essential for the regulation mediated by CaM. Overexpression of the mutant CaM1234, which loses the calcium binding ability of all four EF hands, facilitates Kv7.4 activation by accelerating activation kinetics and shifting the voltage dependence of activation leftwards. The single mutant CaM3, which loses the calcium binding ability of the EF3, phenocopies facilitating effects of CaM1234 on Kv7.4 activation. Kv7.4 channels co-expressed with wild-type (WT) CaM show inhibited activation when intracellular calcium levels increase, while Kv7.4 channels co-expressed with CaM1234 or CaM3 are insensitive to calcium. Mutations C156A, C157A, C158V, R159, and R161A, which are located within the Kv7.4 S2–S3 loop, dramatically facilitate activation of Kv7.4 channels co-expressed with WT CaM but have no effect on activation of Kv7.4 channels co-expressed with CaM3, indicating that these five mutations decrease the inhibitory effect of Ca2+/CaM. The double mutation C156A/R159A decreases Ca2+/CaM binding and completely abolishes CaM-mediated calcium-dependent regulation of Kv7.4 activation. Taken together, our results provide mechanistic insights into CaM regulation of Kv7.4 activation and highlight the crucial role of the Kv7.4 S2–S3 loop in CaM regulation.

Familial neonatal seizures caused by the Kv7.3 selectivity filter mutation T313I.

ObjectiveA spectrum of seizure disorders is linked to mutations in Kv7.2 and Kv7.3 channels. Linking functional effects of identified mutations to their clinical presentation requires ongoing characterization of newly identified variants. In this study, we identified and functionally characterized a previously unreported mutation in the selectivity filter of Kv7.3.MethodsNext‐generation sequencing was used to identify the Kv7.3[T313I] mutation in a family affected by neonatal seizures. Electrophysiological approaches were used to characterize the functional effects of this mutation on ion channels expressed in Xenopus laevis oocytes.ResultsSubstitution of residue 313 from threonine to isoleucine (Kv7.3[T313I]) likely disrupts a critical intersubunit hydrogen bond. Characterization of the mutation in homomeric Kv7.3 channels demonstrated a total loss of channel function. Assembly in heteromeric channels (with Kv7.2) leads to modest suppression of total current when expressed in Xenopus laevis oocytes. Using a Kv7 activator with distinct effects on homomeric Kv7.2 vs heteromeric Kv7.2/Kv7.3 channels, we demonstrated that assembly of Kv7.2 and Kv7.3[T313I] generates functional channels.SignificanceBiophysical and clinical effects of the T313I mutation are consistent with Kv7.3 mutations previously identified in cases of pharmacoresponsive self‐limiting neonatal epilepsy. These findings expand our description of functionally characterized Kv7 channel variants and report new methods to distinguish molecular mechanisms of channel mutations.

Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy

Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in Kv7.2 and Kv7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of Kv7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of Kv7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP2), and our molecular dynamics simulation suggests PIP2 interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in Kv7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of Kv7-associated EE.

Reduced axonal surface expression and phosphoinositide sensitivity in Kv7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy

Neuronal Kv7/KCNQ channels are voltage-gated potassium channels composed of Kv7.2/KCNQ2 and Kv7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in Kv7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of Kv7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to Kv7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP2), revealing novel PIP2 binding residues. While these mutations disrupted Kv7 function to suppress excitability, hyperexcitability was observed in neurons expressing Kv7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in Kv7 channels by severely reducing their CaM binding, K+ currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of Kv7.2, whereas the presence of Kv7.3 blocked this degradation. Furthermore, expression of Kv7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of Kv7.2 cause abnormal Kv7 expression and function by disrupting Kv7.2 binding to CaM and/or modulation by PIP2. We propose that such multiple Kv7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.

D242N, a KV7.1 LQTS Mutation Uncovers a KEY Residue for IKS Voltage Dependence

KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most important repolarizing currents of the cardiac action potential. Its relevance is underscored by the identification of more than 500 mutations in KV7.1 and, at least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this study was to characterize the biophysical and cellular consequences of the D242N KV7.1 mutation associated with the LQTS. The mutation is located in the S4 transmembrane segment, within the voltage sensor of the KV7.1 channel, disrupting the conserved charge balance of this region. Perforated patch-clamp experiments show that, unexpectedly, the mutation did not disrupt the voltage-dependent activation but it removed the inactivation and slowed the activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface protein and co-immunoprecipitation experiments revealed that neither plasma membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized potentials (+50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical phenotype of the LQTS patients carrying the D242N mutation is due to impaired action potential adaptation to exercise and, in particular, to increase in heart rate. Moreover, our data identify D242 aminoacidic position as a potential residue involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel. Funded by SAF2013-45800-R, SAF2016-75021-R, BFU2014-54928-R, RIC RD12/0042/0019 and CIBER CB/11/00222.

Mutations in Kv7.5 Channels Associated with Intellectual Disability or Epileptic Encephalopathy

Mutations in Kv7.5 Channels Associated with Intellectual Disability or Epileptic Encephalopathy

D242N, a KV7.1 LQTS mutation uncovers a key residue for IKs voltage dependence

KV7.1 and KCNE1 co-assemble to give rise to the IKs current, one of the most important repolarizing currents of the cardiac action potential. Its relevance is underscored by the identification of >500 mutations in KV7.1 and, at least, 36 in KCNE1, that cause Long QT Syndrome (LQTS). The aim of this study was to characterize the biophysical and cellular consequences of the D242N KV7.1 mutation associated with the LQTS. The mutation is located in the S4 transmembrane segment, within the voltage sensor of the KV7.1 channel, disrupting the conserved charge balance of this region. Perforated patch-clamp experiments show that, unexpectedly, the mutation did not disrupt the voltage-dependent activation but it removed the inactivation and slowed the activation kinetics of D242N KV7.1 channels. Biotinylation of cell-surface protein and co-immunoprecipitation experiments revealed that neither plasma membrane targeting nor co-assembly between KV7.1 and KCNE1 was altered by the mutation. However, the association of D242N KV7.1 with KCNE1 strongly shifted the voltage dependence of activation to more depolarized potentials (+50mV), hindering IKs current at physiologically relevant membrane potentials. Both functional and computational analysis suggest that the clinical phenotype of the LQTS patients carrying the D242N mutation is due to impaired action potential adaptation to exercise and, in particular, to increase in heart rate. Moreover, our data identify D242 aminoacidic position as a potential residue involved in the KCNE1-mediated regulation of the voltage dependence of activation of the KV7.1 channel.

Competition of calcified calmodulin N lobe and PIP2 to an LQT mutation site in Kv7.1 channel

Voltage-gated potassium 7.1 (Kv7.1) channel and KCNE1 protein coassembly forms the slow potassium current IKS that repolarizes the cardiac action potential. The physiological importance of the IKS channel is underscored by the existence of mutations in human Kv7.1 and KCNE1 genes, which cause cardiac arrhythmias, such as the long-QT syndrome (LQT) and atrial fibrillation. The proximal Kv7.1 C terminus (CT) binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP2), but the role of CaM in channel function is still unclear, and its possible interaction with PIP2 is unknown. Our recent crystallographic study showed that CaM embraces helices A and B with the apo C lobe and calcified N lobe, respectively. Here, we reveal the competition of PIP2 and the calcified CaM N lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor an LQT mutation. Protein pulldown, molecular docking, molecular dynamics simulations, and patch-clamp recordings indicate that residues K526 and K527 in Kv7.1 helix B form a critical site where CaM competes with PIP2 to stabilize the channel open state. Data indicate that both PIP2 and Ca2+-CaM perform the same function on IKS channel gating by producing a left shift in the voltage dependence of activation. The LQT mutant K526E revealed a severely impaired channel function with a right shift in the voltage dependence of activation, a reduced current density, and insensitivity to gating modulation by Ca2+-CaM. The results suggest that, after receptor-mediated PIP2 depletion and increased cytosolic Ca2+, calcified CaM N lobe interacts with helix B in place of PIP2 to limit excessive IKS current inhibition.

Identification of Residues Important for the PUFA Sensitivity of IKs

Kv7.1 and the β-subunit KCNE1 together form the cardiac IKs channel. IKs has an important role in the cardiac repolarization. In a previous study, we showed that polyunsaturated fatty acids (PUFAs) activate the Kv7.1 channel expressed in Xenopus oocytes via an electrostatic mechanism. However, the pharmacological effect was abolished when Kv7.1 was co-expressed with KCNE1. We suggested that KCNE1 promotes protonation of the PUFAs which thereby lose their charge. PUFAs regained their pharmacological effect if pH of the extracellular solution was increased. How KCNE1 promotes protonation of the PUFAs is however unknown. We hypothesise that charged amino acids in the IKs channel change the local pH in the binding site of PUFA. Plausible amino acids to mediate this effect could be in the extracellular part of KCNE1 or that KCNE1 induces a conformational change in Kv7.1 which moves charged amino acids in Kv7.1 close to the PUFA. To determine the mechanism for KCNE1-induced protonation we use a mutagenesis approach where we primarily focus on acidic and basic residues. We also study how different KCNE subunits affect the sensitivity of Kv7.1 to PUFA. We report that deletion of the extracellular part of KCNE1 does not restore the PUFA sensitivity of IKs channels, suggesting that the extracellular part of KCNE1 is not responsible for the protonation of PUFAs. In contrast, specific point mutations in Kv7.1 alter the pharmacological sensitivity of IKs to PUFA. This suggests that residues in Kv7.1 are important for the PUFA sensitivity of IKs. An understanding of how KCNE1 changes the pharmacological sensitivity of IKs is important for the development of drugs that target IKs.

A Structural Characterization of the Kv7.2-Kv7.3 M Channel Proximal C-Terminus/Cam Complex

The Kv7 (KCNQ) family plays major roles in fine-tuning neuronal and cardiomyocyte excitability by reducing firing frequency and controlling repolarization. Kv7 channels have a unique intracellular C-terminus (CT) bound constitutively by calmodulin (CaM), responsible for tetramerization, trafficking, and gating properties. The CT contains four helices, dubbed A through D. CaM embraces the proximal CT, comprised of anti-parallel helices A and B. An X-ray structure of Kv7.1-CT/CaM showed that the CaM C-lobe in apo form binds helix A, while Ca2+/N-lobe binds helix B. Concatameric channels demonstrated that CaM binds both helices within the same Kv7.1 subunit. Kv7.2 and Kv7.3 are the predominant members found in neural tissues. Together they form the heterotetrameric M channel, named after the IM current it conducts. IM yields larger current amplitudes compared to currents generated by Kv7.2 homomers. Kv7.3 channel conductance is difficult to distinguish from background level and may not exist as a homomeric channel in vivo. Mutations in Kv7.2 or Kv7.3 cause neonatal epileptic encephalopathies and myokymia.We characterized Kv7.2, Kv7.3 and chimeric Kv7.3 helix A-Kv7.2 helix B (Q3A-Q2B) proximal CT/CaM complexes by light-scattering and SAXS at various Ca2+ concentrations. We tested the chimeric channel and found it to be functional. We subsequently determined the crystal structure of the Q3A-Q2B/CaM complex at high [Ca2+] to 2.0 A resolution. Our results indicate unique interactions responsible for the inter-subunit pairing of Q3A and Q2B while suggesting that Q3A-Q2B/CaM binding may be similar to that observed for Kv7.1-CT/CaM and unlike the model suggested for Kv7.4-CT/CaM. The Q3A-Q2B/CaM structure can be used to rationalize various Kv7.2 channelopathic mutants. Other implications for M-channel structure-function will be discussed.

Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin

Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K+ channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca2+). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies.

Early-onset epileptic encephalopathy caused by gain-of-function mutations in the voltage sensor of Kv7.2 and Kv7.3 potassium channel subunits.

Mutations in Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) genes, encoding for voltage-gated K(+) channel subunits underlying the neuronal M-current, have been associated with a wide spectrum of early-onset epileptic disorders ranging from benign familial neonatal seizures to severe epileptic encephalopathies. The aim of the present work has been to investigate the molecular mechanisms of channel dysfunction caused by voltage-sensing domain mutations in Kv7.2 (R144Q, R201C, and R201H) or Kv7.3 (R230C) recently found in patients with epileptic encephalopathies and/or intellectual disability. Electrophysiological studies in mammalian cells transfected with human Kv7.2 and/or Kv7.3 cDNAs revealed that each of these four mutations stabilized the activated state of the channel, thereby producing gain-of-function effects, which are opposite to the loss-of-function effects produced by previously found mutations. Multistate structural modeling revealed that the R201 residue in Kv7.2, corresponding to R230 in Kv7.3, stabilized the resting and nearby voltage-sensing domain states by forming an intricate network of electrostatic interactions with neighboring negatively charged residues, a result also confirmed by disulfide trapping experiments. Using a realistic model of a feedforward inhibitory microcircuit in the hippocampal CA1 region, an increased excitability of pyramidal neurons was found upon incorporation of the experimentally defined parameters for mutant M-current, suggesting that changes in network interactions rather than in intrinsic cell properties may be responsible for the neuronal hyperexcitability by these gain-of-function mutations. Together, the present results suggest that gain-of-function mutations in Kv7.2/3 currents may cause human epilepsy with a severe clinical course, thus revealing a previously unexplored level of complexity in disease pathogenetic mechanisms.

Cellular mechanisms of mutations in Kv7.1: auditory functions in Jervell and Lange-Nielsen syndrome vs. Romano–Ward syndrome

As a result of cell-specific functions of voltage-activated K+ channels, such as Kv7.1, mutations in this channel produce profound cardiac and auditory defects. At the same time, the massive diversity of K+ channels allows for compensatory substitution of mutant channels by other functional channels of their type to minimize defective phenotypes. Kv7.1 represents a clear example of such functional dichotomy. While several point mutations in the channel result in a cardio-auditory syndrome called Jervell and Lange-Nielsen syndrome (JLNS), about 100-fold mutations result in long QT syndrome (LQTS) denoted as Romano–Ward syndrome (RWS), which has an intact auditory phenotype. To determine whether the cellular mechanisms for the diverse phenotypic outcome of Kv7.1 mutations, are dependent on the tissue-specific function of the channel and/or specialized functions of the channel, we made series of point mutations in hKv7.1 ascribed to JLNS and RWS. For JLNS mutations, all except W248F yielded non-functional channels when expressed alone. Although W248F at the end of the S4 domain yielded a functional current, it underwent marked inactivation at positive voltages, rendering the channel non-functional. We demonstrate that by definition, none of the JLNS mutants operated in a dominant negative (DN) fashion. Instead, the JLNS mutants have impaired membrane trafficking, trapped in the endoplasmic reticulum (ER) and Cis-Golgi. The RWS mutants exhibited varied functional phenotypes. However, they can be summed up as exhibiting DN effects. Phenotypic differences between JLNS and RWS may stem from tissue-specific functional requirements of cardiac vs. inner ear non-sensory cells.

Mutations in the potassium channel subunit KCNE1 are associated with early-onset familial atrial fibrillation

BackgroundAtrial fibrillation (AF) is the most common arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutations in KV7.1, the pore-forming α-subunit of the IKs channel, have been associated with AF. We hypothesized that early-onset lone AF is associated with mutations in the IKs channel regulatory subunit KCNE1.MethodsIn 209 unrelated early-onset lone AF patients (< 40 years) the entire coding sequence of KCNE1 was bidirectionally sequenced. We analyzed the identified KCNE1 mutants electrophysiologically in heterologous expression systems.ResultsTwo non-synonymous mutations G25V and G60D were found in KCNE1 that were not present in the control group (n = 432 alleles) and that have not previously been reported in any publicly available databases or in the exom variant server holding exom data from more than 10.000 alleles. Proband 1 (female, age 45, G25V) had onset of paroxysmal AF at the age of 39 years. Proband 2 (G60D) was diagnosed with lone AF at the age of 33 years. The patient has inherited the mutation from his mother, who also has AF. Both probands had no mutations in genes previously associated with AF. In heterologous expression systems, both mutants showed significant gain-of-function for IKs both with respect to steady-state current levels, kinetic parameters, and heart rate-dependent modulation.ConclusionsMutations in KV7.1 leading to gain-of-function of IKs current have previously been described in lone AF, yet this is the first time a mutation in the beta-subunit KCNE1 is associated with the disease. This finding further supports the hypothesis that increased potassium current enhances AF susceptibility.

Kv7 Channels Can Function without Constitutive Calmodulin Tethering

M-channels are voltage-gated potassium channels composed of Kv7.2-7.5 subunits that serve as important regulators of neuronal excitability. Calmodulin binding is required for Kv7 channel function and mutations in Kv7.2 that disrupt calmodulin binding cause Benign Familial Neonatal Convulsions (BFNC), a dominantly inherited human epilepsy. On the basis that Kv7.2 mutants deficient in calmodulin binding are not functional, calmodulin has been defined as an auxiliary subunit of Kv7 channels. However, we have identified a presumably phosphomimetic mutation S511D that permits calmodulin-independent function. Thus, our data reveal that constitutive tethering of calmodulin is not required for Kv7 channel function.

Gating Currents from Neuronal K V7 Channels Carrying BFNS-Causing Mutations in the S 4 Segment of the Voltage Sensing Domain

Kv7 genes encode for channel voltage-gated potassium subunits with inhibitory function on electrical excitability. Mutations in Kv7.1 cause one form of long QT syndrome, whereas Kv7.4 mutations underlie a rare form of slowly progressive deafness; finally, mutations in Kv7.2/Kv7.3 genes have been identified in families affected by Benign Familial Neonatal Seizures (BFNS) and/or peripheral nerve hyperexcitability (PNH). Disease-causing mutations often affect residues in the voltage-sensing domain of Kv7 subunits and modify the gating properties of the macroscopic currents carried by these channels. To achieve a more detailed functional analysis of these gating changes, we previously recorded the gating currents from the neuronal Kv7.4 channel, using the cut-open vaseline gap technique. In the present work, we have characterized the ionic and gating currents from homomeric Kv7.4 channels carrying mutations homologous to BFNS-causing mutations in Kv7.2 (R213Q/W, D218G and R219W), as well as the non-BFNS mutant R219Q (Kv7.4 numbering). Channels carrying the D218G, R219Q or the R219W mutations in the C-terminal part of S4 show the following salient properties: +30 mV shift of the G/V curves and faster activation/deactivation kinetics. Gating currents from these channels showed: gating charge conservation between QON and QOFF, slight (5-13 mV) right-shift of the Q/V curves, and a fast QOFF decay with no rising phase. Neutralization of R213 caused a G/V right-shift associated to slower activation/deactivation channel kinetics. Interestingly, upon pore current blockade, in Kv7.4 channels carrying the R213Q/W mutations causing PNH, a persistent outward current was recorded that increases on depolarization, indicating a gating pore leak in the active state of the sensor. These data may help elucidate the molecular pathogenesis of BFNS and associated diseases.Supported by Telethon GP07125 (MT), E-Rare JTC 2007 (MRC), and NIH GM30376 (FB).

Gating currents from neuronal Kv7 channels

The Kv7 family of voltage-gated K+ channels consists of five members (Kv7.1- Kv7.5), each showing specific tissue distribution and physiological role. Mutations in Kv7.1 cause the long QT syndrome, whereas Kv7.4 mutations underlie a rare form of slowly progressive deafness (DFNA2); finally, mutations in Kv7.2 and Kv7.3 genes have been identified in families affected by Benign Familial Neonatal Seizures. Disease-causing mutations often affect residues in the voltage-sensing domain of Kv7 subunits. In the present study, we have characterized the ionic and gating current of homomeric neuronally-expressed Kv7 channels (Kv7.2-Kv7.5) using the cut-open oocyte voltage clamp. The ionic and gating current were recorded at 18°C and also at 28°C to speed up gating charge movement. Increasing the recording temperature from 18°C to 28°C caused an accelerated activation/deactivation kinetics of the ionic currents in all homomeric Kv7 channels (the Q10 for the activation kinetics at 0 mV was 3.8, 4.1, 8.3, and 2.8 for Kv7.2, Kv7.3, Kv7.4 and Kv7.5). Moreover, currents carried by Kv7.4 channels (and less so Kv7.2) also showed a significant increase in their maximal value. Gating currents were only resolved in Kv7.4 and Kv7.5 channels, possibly because of an higher membrane channel density; ON gating charges at saturated potential (+40mV) amounted to 1.34±0.34 (Kv7.4) and 0.79±0.20 nC (Kv7.5). At 28°C, Kv7.4 gating currents had the following salient properties: 1) similar time integral of ON and OFF, indicating charge conservation; 2) a left-shift in the V1/2 of the ON gating charge when compared to ionic currents; 3) a rising phase in the OFF gating charge after depolarizations to values >0 mV. These results represent the first description of Kv7.4 and Kv7.5 gating currents and may help to clarify the molecular consequence of disease-causing mutations affecting channel gating. Supported by NIH GM30376 and Telethon GGP07125.