Family Of Voltage-gated Potassium Channels Research Articles

Mono-allelic KCNB2 variants lead to a neurodevelopmental syndrome caused by altered channel inactivation

Ion channels mediate voltage fluxes or action potentials that are central to the functioning of excitable cells such as neurons. The KCNB family of voltage-gated potassium channels (Kv) consists of two members (KCNB1 and KCNB2) encoded by KCNB1 and KCNB2, respectively. These channels are major contributors to delayed rectifier potassium currents arising from the neuronal soma which modulate overall excitability of neurons. In this study, we identified several mono-allelic pathogenic missense variants in KCNB2, in individuals with a neurodevelopmental syndrome with epilepsy and autism in some individuals. Recurrent dysmorphisms included a broad forehead, synophrys, and digital anomalies. Additionally, we selected three variants where genetic transmission has not been assessed, from two epilepsy studies, for inclusion in our experiments. We characterized channel properties of these variants by expressing them in oocytes of Xenopus laevis and conducting cut-open oocyte voltage clamp electrophysiology. Our datasets indicate no significant change in absolute conductance and conductance-voltage relationships of most disease variants as compared to wild type (WT), when expressed either alone or co-expressed with WT-KCNB2. However, variants c.1141A>G (p.Thr381Ala) and c.641C>T (p.Thr214Met) show complete abrogation of currents when expressed alone with the former exhibiting a left shift in activation midpoint when expressed alone or with WT-KCNB2. The variants we studied, nevertheless, show collective features of increased inactivation shifted to hyperpolarized potentials. We suggest that the effects of the variants on channel inactivation result in hyper-excitability of neurons, which contributes to disease manifestations.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Mutations affecting electro-mechanical coupling in KCNQ channels alter kinetics and voltage dependence of activation.

The KCNQ voltage-gated potassium channel family serves multiple physiological functions, from regulating heartbeat (by KCNQ1) to controlling neuronal excitability (by KCNQ2-5). In KCNQ1 channels, two steps of voltage sensor activation lead to two functionally distinct open states, the intermediate-open (IO) and activated-open (AO), which define the gating, pharmacology, and physiology of KCNQ1. However, whether all KCNQ channels share the same mechanism is unclear. Here, we provide functional evidence that KCNQ2/3 only conducts at the AO state, but the electro-mechanical (E-M) coupling mechanism is conserved between KCNQ1 and KCNQ2/3. We demonstrate that this conserved E-M coupling can be compromised by select epilepsy-linked mutations in KCNQ2/3 and also can be effectively targeted by the antiepileptic prototype drug retigabine, suggesting E-M coupling as a therapeutic target for epilepsy treatment. Interestingly, KCNQ1 and KCNQ2/3 exhibit distinct kinetics and voltage dependence of activation for the AO state. While these differences may have important implications for the primary physiological roles of KCNQ2/3 (modulation of neuronal excitability) versus KCNQ1 (repolarization of cardiac action potential), it remains unclear where they originate from. Here, we find that analogous mutations in the distal S6 of KCNQ1 and KCNQ2 both affect the AO state E-M coupling and lead to delays in current activation kinetics and right shifts in voltage dependence of activation. KCNQ2 mutations near this location are also found to be associated with epileptic encephalopathy. These results further support a common regulatory mechanism for KCNQ currents via E-M coupling and indicate the physiological relevance of altering AO state activation properties in KCNQ channels.

Intact Drosophila central nervous system cellular quantitation reveals sexual dimorphism.

Establishing with precision the quantity and identity of the cell types of the brain is a prerequisite for a detailed compendium of gene and protein expression in the central nervous system (CNS). Currently, however, strict quantitation of cell numbers has been achieved only for the nervous system of Caenorhabditis elegans. Here, we describe the development of a synergistic pipeline of molecular genetic, imaging, and computational technologies designed to allow high-throughput, precise quantitation with cellular resolution of reporters of gene expression in intact whole tissues with complex cellular constitutions such as the brain. We have deployed the approach to determine with exactitude the number of functional neurons and glia in the entire intact larval Drosophila CNS, revealing fewer neurons and more glial cells than previously predicted. We also discover an unexpected divergence between the sexes at this juvenile developmental stage, with the female CNS having significantly more neurons than that of males. Topological analysis of our data establishes that this sexual dimorphism extends to deeper features of CNS organisation. We additionally extended our analysis to quantitate the expression of voltage-gated potassium channel family genes throughout the CNS and uncover substantial differences in abundance. Our methodology enables robust and accurate quantification of the number and positioning of cells within intact organs, facilitating sophisticated analysis of cellular identity, diversity, and gene expression characteristics.

Pentameric assembly of the Kv2.1 tetramerization domain.

The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristics of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily lacking a representative structure of the T1 domain is the Kv2 family. Here, X-ray crystallography was used to solve the structure of the human Kv2.1 T1 domain. The structure is similar to those of other T1 domains, but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer, as determined by inline SEC-MALS-SAXS and negative-stain electron microscopy. The Kv2.1 T1-T1 interface involves electrostatic interactions, including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. It is shown that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER-plasma membrane contacts.

Cannabidiol differentially modulates Kv7 channelopathy-related phenotypes

Mutations in members of voltage-gated potassium channel family 7 (Kv7) modify electrophysiological properties and cause distinct clinical presentations. Mutations of the M-channel (Kv7.2/3 heterotetramer) underlying the neuronal M-current cause benign familial neonatal epilepsy (BFNE). Mutations in the Kv7.1 or KCNE1 subunit of the Kv7.1/KCNE1 heteromultimer underlying the ventricular repolarization current IKS cause Long-(L)QT-syndrome. The phytocannabinoid cannabidiol (CBD) is an FDA-approved anticonvulsant.

How to Grow a Tree: Plant Voltage-Dependent Cation Channels in the Spotlight of Evolution.

Phylogenetic analysis can be a powerful tool for generating hypotheses regarding the evolution of physiological processes. Here, we provide an updated view of the evolution of the main cation channels in plant electrical signalling: the Shaker family of voltage-gated potassium channels and the two-pore cation (K+) channel (TPC1) family. Strikingly, the TPC1 family followed the same conservative evolutionary path as one particular subfamily of Shaker channels (Kout) and remained highly invariant after terrestrialisation, suggesting that electrical signalling was, and remains, key to survival on land. We note that phylogenetic analyses can have pitfalls, which may lead to erroneous conclusions. To avoid these in the future, we suggest guidelines for analyses of ion channel evolution in plants.

SMIT (Sodium-Myo-Inositol Transporter) 1 Regulates Arterial Contractility Through the Modulation of Vascular Kv7 Channels.

The SMIT1 (sodium:myo-inositol transporter 1) regulates myo-inositol movement into cells and responses to hypertonic stimuli. Alteration of myo-inositol levels has been associated with several diseases, including hypertension, but there is no evidence of a functional role of SMIT1 in the vasculature. Recent evidence showed that in the nervous system SMIT1 interacted and modulated the function of members of the Kv7 family of voltage-gated potassium channels, which are also expressed in the vasculature where they regulate arterial contractility. Therefore, in this study, we evaluated whether SMIT1 was functionally relevant in arterial smooth muscle. Approach and Results: Immunofluorescence and polymerase chain reaction experiments revealed that SMIT1 was expressed in rat renal and mesenteric vascular smooth muscle cells. Isometric tension recordings showed that incubation of renal arteries with raffinose and myo-inositol (which increases SMIT1 expression) reduced the contractile responses to methoxamine, an effect that was abolished by preincubation with the pan-Kv7 blocker linopirdine and by molecular knockdown of Kv7.4 and Kv7.5. Knockdown of SMIT1 increased the contraction of renal arteries induced by methoxamine, impaired the response to the Kv7.2-Kv7.5 activator ML213 but did not interfere with the relaxant responses induced by openers of other potassium channels. Proximity ligation assay showed that SMIT1 interacted with heteromeric channels formed by Kv7.4 and Kv7.5 proteins in both renal and mesenteric vascular smooth muscle cells. Patch-clamp experiments showed that incubation with raffinose plus myo-inositol increased Kv7 currents in vascular smooth muscle cells. SMIT1 protein is expressed in vascular smooth muscle cells where it modulates arterial contractility through an association with Kv7.4/Kv7.5 heteromers.

Heteromeric Channels Formed From Alternating Kv7.4 and Kv7.5 α-Subunits Display Biophysical, Regulatory, and Pharmacological Characteristics of Smooth Muscle M-Currents.

Smooth muscle cells of the vasculature, viscera, and lungs generally express multiple α-subunits of the Kv7 voltage-gated potassium channel family, with increasing evidence that both Kv7.4 and Kv7.5 can conduct “M-currents” that are functionally important for the regulation of smooth muscle contractility. Although expression systems demonstrate that functional channels can form as homomeric tetramers of either Kv7.4 or Kv7.5 α-subunits, there is evidence that heteromeric channel complexes, containing some combination of Kv7.4 and Kv7.5 α-subunits, may represent the predominant configuration natively expressed in some arterial myocytes, such as rat mesenteric artery smooth muscle cells (MASMCs). Our previous work has suggested that Kv7.4/Kv7.5 heteromers can be distinguished from Kv7.4 or Kv7.5 homomers based on their biophysical, regulatory, and pharmacological characteristics, but it remains to be determined how Kv7.4 and Kv7.5 α-subunits combine to produce these distinct characteristics. In the present study, we constructed concatenated dimers or tetramers of Kv7.4 and Kv7.5 α-subunits and expressed them in a smooth muscle cell line to determine if a particular α-subunit configuration can exhibit the features previously reported for natively expressed Kv7 currents in MASMCs. Several unique characteristics of native smooth muscle M-currents were reproduced under conditions that constrain channel formation to a Kv7.4:Kv7.5 stoichiometry of 2:2, with alternating Kv7.4 and Kv7.5 α-subunits within a tetrameric structure. Although other subunit arrangements/combinations are not ruled out, the findings provide new insights into the oligomerization of α-subunits and the ways in which Kv7.4/Kv7.5 subunit assembly can affect smooth muscle signal transduction and pharmacological responses to Kv7 channel modulating drugs.

Efficacy, potency and kinetics of activation process of Kv7.4 and Kv7.5 channel with novel pharmacological activators targeted for Lower Urinary Tract Syndrome (LUTS)

Among several types of Kv7 (KCNQ) voltage gated potassium channel family, Kv7.4 (KCNQ4) and Kv7.5 (KCNQ5) channels show prominent expression in smooth muscle, cardiac tissues and vasculature. In general, activation of the channel would mediate strong efflux of potassium and lead membrane potential to hyperpolarization, though the extent may depend on established potassium concentration gradient and membrane potential. Once membrane potential is hyperpolarized, it would decrease the excitability of excitable cells. Decreased excitability may reduce calcium spikes and Ca‐CaM mediated activation of myosin light chain kinase (MLCK) in smooth muscle cells. Recently, it was reported that Kv7.4 channels are highly expressed in corpus cavernosum smooth muscle cells and Kv7.5 channels in prostate smooth muscle cells, respectively. Since smooth muscle relaxation of corpus cavernosum and smooth muscle tension in prostate are in the center of pathophysiology of erectile dysfunction and LUTS, we examined the effect of novel Kv7.4 and Kv7.5 activators to the channel activation process.In HEK293 cells expressing human Kv7.4 or Kv7.5 channels, we performed whole‐cell patch clamp technique with two different voltage protocols in order to measure specific activity of the channel. For kinetic analysis of the activation process, voltage steps from –100 mV to +100 mV with 10 mV of step interval was applied. For each step, pulse duration was 3 seconds for activation process of Kv7.4 and Kv7.5 channels is rather slow. Intersweep duration was 10 seconds and holding potential was –80 mV. For measurement of time‐dependent action of the drug, 10 seconds single step with +50 mV was applied in every 30s with holding potential of –80 mV. The composition of internal solution is as following; 120 KCl, 5.37 CaCl2, 1.75 MgCl2, 10 EGTA, 10 HEPES, 4 ATP, 0.2 GTP. pH was adjusted to 7.3 using KOH and [Ca2+]free = 100 nM. Normal tyrode solution was used for external solution and drugs stocked with DMSO was diluted to corresponding concentrations with the Normal tyrode solution.We have found that URO‐K10, a novel activator, increases not only maximum conductance of the channel but also time constant of activation gate. In comparison to positive control, ML‐213, a well‐known Kv7.4 channel activator, URO‐K10 showed significant supremacy. EC50 of URO‐K10 was 0.304 ± 0.058 μM for Kv7.4 channel which is smaller than EC50 of ML‐213 (0.8 ± 0.3 μM). URO‐K10 not only increased the maximum conductance of the channel but also left‐shifted activation curve in terms of Boltzmann distribution. Half‐maximal voltage (V1/2) of Kv7.4 channel without any of the drugs was −43.03 mV whereas V1/2 of URO‐K10 activated channel was −67.65 mV, which is near the resting membrane potential of smooth muscle cells. ML‐213 activated channels showed V1/2 of −51.91 mV.Support or Funding InformationThis work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information & Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Jung Eun Lee was supported by the BK plus program from the MSIP.

Calmodulin: A Multitasking Protein in Kv7.2 Potassium Channel Functions.

The ubiquitous calcium transducer calmodulin (CaM) plays a pivotal role in many cellular processes, regulating a myriad of structurally different target proteins. Indeed, it is unquestionable that CaM is the most relevant transductor of calcium signals in eukaryotic cells. During the last two decades, different studies have demonstrated that CaM mediates the modulation of several ion channels. Among others, it has been indicated that Kv7.2 channels, one of the members of the voltage gated potassium channel family that plays a critical role in brain excitability, requires CaM binding to regulate the different mechanisms that govern its functions. The purpose of this review is to provide an overview of the most recent advances in structure–function studies on the role of CaM regulation of Kv7.2 and the other members of the Kv7 family.

Kv10.1 potassium channel: from the brain to the tumors.

The KCNH1 gene encodes the Kv10.1 (Eag1) ion channel, a member of the EAG (ether-à-go-go) family of voltage-gated potassium channels. Recent studies have demonstrated that KCHN1 mutations are implicated in Temple-Baraitser and Zimmermann-Laband syndromes and other forms of developmental deficits that all present with mental retardation and epilepsy, suggesting that Kv10.1 might be important for cognitive development in humans. Although the Kv10.1 channel is mainly expressed in the mammalian brain, its ectopic expression occurs in 70% of human cancers. Cancer cells and tumors expressing Kv10.1 acquire selective advantages that favor cancer progression through molecular mechanisms that involve several cellular pathways, indicating that protein-protein interactions may be important for Kv10.1 influence in cell proliferation and tumorigenesis. Several studies on transcriptional and post-transcriptional regulation of Kv10.1 expression have shown interesting mechanistic insights about Kv10.1 role in oncogenesis, increasing the importance of identifying the cellular factors that regulate Kv10.1 expression in tumors.

Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25–27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca2+i) through binding of Ca2+-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca2+-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca2+i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca2+i. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca2+i. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca2+i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca2+-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.

Structure of human dipeptidyl peptidase 10 (DPPY): a modulator of neuronal Kv4 channels.

The voltage-gated potassium channel family (Kv) constitutes the most diverse class of ion channels in the nervous system. Dipeptidyl peptidase 10 (DPP10) is an inactive peptidase that modulates the electrophysiological properties, cell-surface expression and subcellular localization of voltage-gated potassium channels. As a consequence, DPP10 malfunctioning is associated with neurodegenerative conditions like Alzheimer and fronto-temporal dementia, making this protein an attractive drug target. In this work, we report the crystal structure of DPP10 and compare it to that of DPP6 and DPP4. DPP10 belongs to the S9B serine protease subfamily and contains two domains with two distinct folds: a β-propeller and a classical α/β-hydrolase fold. The catalytic serine, however, is replaced by a glycine, rendering the protein enzymatically inactive. Difference in the entrance channels to the active sites between DPP10 and DPP4 provide an additional rationale for the lack of activity. We also characterize the DPP10 dimer interface focusing on the alternative approach for designing drugs able to target protein-protein interactions.

Evolutionary Origins of the Shaker Family of Voltage-Gated Potassium Channels

The Shaker family of voltage-gated K+ channels in bilaterians consists of four closely related gene subfamilies (Shaker, Shab, Shaw and Shal) that regulate neuronal excitability in diverse ways. We examined the evolutionary origins of the gene family by characterizing Shaker channels in early branching metazoan lineages. Shaker family channels can be definitively identified by the combination of a voltage-dependent potassium channel core and a cytosolic domain (T1) that mediates subfamily-specific tetrameric assembly. The Shaker, Shab, Shaw and Shal subfamilies are present in cnidarians, a close sister group to the bilaterians. We show that all four subfamilies are highly conserved on a functional level in the sea anemone Nematostella vectensis, an emerging cnidarian model organism. The Nematostella Shaker, Shal and Shaw subfamilies include cnidarian-specific expansions of regulatory subunits that extend functional diversity through the formation of heteromeric channels. We were unable to identify Shaker family genes in choanoflagellates or sponges, but found over 40 Shaker family genes in a ctenophore (Mnemiopsis leidyi). Ctenophores are the earliest branching metazoans with a nervous system. Phylogenetic analysis indicates that the ctenophore Shaker family expanded independently and diverged prior to emergence of the Shaker, Shab, Shal and Shaw subfamilies. The phylogeny and functional analysis support Shaker as the oldest of these subfamilies. Two Mnemiopsis Shaker family subunits heteromerize with Nematostella and mouse Shaker subfamily channels, but not with Shab, Shaw or Shal. Thus the subunit interface of Shaker subfamily may retain ancestral features. Our results indicate that the Shaker family is metazoan-specific and was present by the time neurons evolved. However, the Shaker, Shab, Shal and Shaw subfamilies evolved in the cnidarian/bilaterian lineage long after the evolution of the first nervous systems.

Homologous Domains Mediate Distinct Gating Functions in EAG vs. Cyclic-Nucleotide Gated Channels

EAG, ERG and ELK are members of the Ether-a-go-go voltage-gated potassium channel family. Each channel possesses a C-terminal domain that is homologous to the cyclic nucleotide-binding domain (CNBD) of CNG/HCN channels but insensitive to cyclic nucleotides. X-ray crystallography of EAG suggests the side chains of two residues, Y699 and L701, interact in a manner analogous to the two moieties of a cyclic nucleotide in the binding pocket of bona fide CNBDs. Using two-electrode voltage clamp of channel proteins expressed in Xenopus oocytes, we found that alanine substitutions of Y699 and L701 (“AA”) dramatically slowed activation and shifted the g-V curve by +15 mV. Mutagenesis of both residues were required to elicit an effect, as if the “apo” configuration required the loss of both side chains. Thus, the intrinsic ligand of the CNB homology domain (CNBhD) reduces the stability of the closed state relative to the open state. To determine whether the AA mutant phenotype represents a loss of function, we deleted the CNBhD. In contrast to the AA mutant, the deletion gave rise to faster activation kinetics but a more dramatic g-V shift of +40 mV. This finding indicates that, unlike HCN channel, in which the unliganded CNBD mimics the CNBD deletion, the AA mutant phenotype is not a loss of function of the CNBhD. Instead, both the intrinsically-liganded and apo conformations communicate with the gating machinery. The current properties can be recapitulated by a 7-state allosteric model, in which the AA mutant alters multiple transitions during channel gating whereas CNBhD deletion affects only the last opening step. We conclude that the EAG1 CNBhD serves a function mechanistically distinct from those of the corresponding domains in channels gated by cyclic nucleotides despite extensive sequence and structural homology.

Contribution of Kv7 channels to natriuretic peptide mediated vasodilation in normal and hypertensive rats.

The Kv7 family of voltage-gated potassium channels are expressed within the vasculature where they are key regulators of vascular tone and mediate cAMP-linked endogenous vasodilator responses, a pathway that is compromised in hypertension. However, the role of Kv7 channels in non-cAMP-linked vasodilator pathways has not been investigated. Natriuretic peptides are potent vasodilators, which operate primarily through the activation of a cGMP-dependent signaling pathway. This study investigated the putative role of Kv7 channels in natriuretic peptide-dependent relaxations in the vasculature of normal and hypertensive animals. Relaxant responses of rat aorta to both atrial and C-type natriuretic peptides and the nitric oxide donor sodium nitroprusside were impaired by the Kv7 blocker linopirdine (10 μmol/L) but not by the Kv7.1-specific blocker HMR1556 (10 μmol/L) and other K(+) channel blockers. In contrast, only the atrial natriuretic peptide response was sensitive to linopirdine in the renal artery. These Kv7-mediated responses were attenuated in arteries from hypertensive rats. Quantitative polymerase chain reaction showed that A- and B-type natriuretic peptide receptors were expressed at high levels in the aorta and renal artery from normal and spontaneously hypertensive rats. This study provides the first evidence that natriuretic peptide responses are impaired in hypertension and that recruitment of Kv7 channels is a key component of natriuretic peptide-dependent vasodilations.

Vasorelaxant effects of novel Kv 7.4 channel enhancers ML213 and NS15370.

The KCNQ-encoded voltage-gated potassium channel family (Kv 7.1-Kv 7.5) are established regulators of smooth muscle contractility, where Kv 7.4 and Kv 7.5 predominate. Various Kv 7.2-7.5 channel enhancers have been developed that have been shown to cause a vasorelaxation in both rodent and human blood vessels. Recently, two novel Kv 7 channel enhancers have been identified, ML213 and NS15370, that show increased potency, particularly on Kv 7.4 channels. The aim of this study was to characterize the effects of these novel enhancers in different rat blood vessels and compare them with Kv 7 enhancers (S-1, BMS204352, retigabine) described previously. We also sought to determine the binding sites of the new Kv 7 enhancers. Both ML213 and NS15370 relaxed segments of rat thoracic aorta, renal artery and mesenteric artery in a concentration-dependent manner. In the mesenteric artery ML213 and NS15370 displayed EC50 s that were far lower than other Kv 7 enhancers tested. Current-clamp experiments revealed that both novel enhancers, at low concentrations, caused significant hyperpolarization in mesenteric artery smooth muscle cells. In addition, we determined that the stimulatory effect of these enhancers relied on a tryptophan residue located in the S5 domain, which is the same binding site for the other Kv 7 enhancers tested in this study. This study has identified and characterized ML213 and NS15370 as potent vasorelaxants in different blood vessels, thereby highlighting these new compounds as potential therapeutics for various smooth muscle disorders.

Targeted high-throughput sequencing identifies pathogenic mutations in KCNQ4 in two large Chinese families with autosomal dominant hearing loss.

Autosomal dominant non-syndromic hearing loss (ADNSHL) is highly heterogeneous, among them, KCNQ4 is one of the most frequent disease-causing genes. More than twenty KCNQ4 mutations have been reported, but none of them were detected in Chinese mainland families. In this study, we identified a novel KCNQ4 mutation in a five generation Chinese family with 84 members and a known KCNQ4 mutation in a six generation Chinese family with 66 members. Mutation screening of 30 genes for ADNSHL was performed in the probands from thirty large Chinese families with ADNSHL by targeted region capture and high-throughput sequencing. The candidate variants and the co-segregation of the phenotype were verified by polymerase chain reaction (PCR) amplification and Sanger sequencing in all ascertained family members. Then we identified a novel KCNQ4 mutation p.W275R in exon 5 and a known KCNQ4 mutation p.G285S in exon 6 in two large Chinese ADNSHL families segregating with post-lingual high frequency-involved and progressive sensorineural hearing loss. This is the first report of KCNQ4 mutation in Chinese mainland families. KCNQ4, a member of voltage-gated potassium channel family, is likely to be a common gene in Chinese patients with ADNSHL. The results also support that the combination of targeted enrichment and high-throughput sequencing is a valuable molecular diagnostic tool for autosomal dominant hereditary deafness.

Novel Kv7.1-Phosphatidylinositol 4,5-Bisphosphate Interaction Sites Uncovered by Charge Neutralization Scanning

Kv7.1 to Kv7.5 α-subunits belong to the family of voltage-gated potassium channels (Kv). Assembled with the β-subunit KCNE1, Kv7.1 conducts the slowly activating potassium current IKs, which is one of the major currents underlying repolarization of the cardiac action potential. A known regulator of Kv7 channels is the lipid phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 increases the macroscopic current amplitude by stabilizing the open conformation of 7.1/KCNE1 channels. However, knowledge about the exact nature of the interaction is incomplete. The aim of this study was the identification of the amino acids responsible for the interaction between Kv7.1 and PIP2. We generated 13 charge neutralizing point mutations at the intracellular membrane border and characterized them electrophysiologically in complex with KCNE1 under the influence of diC8-PIP2. Electrophysiological analysis of corresponding long QT syndrome mutants suggested impaired PIP2 regulation as the cause for channel dysfunction. To clarify the underlying structural mechanism of PIP2 binding, molecular dynamics simulations of Kv7.1/KCNE1 complexes containing two PIP2 molecules in each subunit at specific sites were performed. Here, we identified a subset of nine residues participating in the interaction of PIP2 and Kv7.1/KCNE1. These residues may form at least two binding pockets per subunit, leading to the stabilization of channel conformations upon PIP2 binding.