Polyunsaturated fatty acid analogues differentially affect cardiac NaV, CaV, and KV channels through unique mechanisms.

The cardiac ventricular action potential depends on several voltage-gated ion channels, including NaV, CaV, and KV channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (NaV, CaV, and KV). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

Structure and physiological function of the human KCNQ1 channel voltage sensor intermediate state.

Voltage-gated ion channels feature voltage sensor domains (VSDs) that exist in three distinct conformations during activation: resting, intermediate, and activated. Experimental determination of the structure of a potassium channel VSD in the intermediate state has previously proven elusive. Here, we report and validate the experimental three-dimensional structure of the human KCNQ1 voltage-gated potassium channel VSD in the intermediate state. We also used mutagenesis and electrophysiology in Xenopus laevisoocytes to functionally map the determinants of S4 helix motion during voltage-dependent transition from the intermediate to the activated state. Finally, the physiological relevance of the intermediate state KCNQ1 conductance is demonstrated using voltage-clamp fluorometry. This work illuminates the structure of the VSD intermediate state and demonstrates that intermediate state conductivity contributes to the unusual versatility of KCNQ1, which can function either as the slow delayed rectifier current (IKs) of the cardiac action potential or as a constitutively active epithelial leak current.

Two-stage electro-mechanical coupling of a KV channel in voltage-dependent activation.

In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro-mechanical (E-M) coupling. However, the spatial and temporal details underlying E-M coupling remain elusive. Here, utilizing KV7.1's unique two open states, we report a two-stage E-M coupling mechanism in voltage-dependent gating of KV7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore of the neighboring subunit to activate conductance. This two-stage hand-and-elbow gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous domain-swapped KV channels.

Reversal of a Treatment-Resistant, Depression-Related Brain State with the Kv7 Channel Opener Retigabine.

Neuroinflammation is associated with increased vulnerability to diverse psychiatric conditions, including treatment-resistant major depressive disorder (MDD). Here we assessed whether high fat diet (HFD) induced neuroinflammation may be suitable to model a treatment-resistant depressive-like brain state in mice. Male and female mice were fed a HFD for 18 weeks, followed by quantitation of glucose tolerance, inflammatory markers of brain tissue (TNFα, IL-6, IL-1ß, Iba-1), neural excitability in the prelimbic cortex (PLC), as well as assessment of emotional reactivity and hedonic behavior in a battery of behavioral tests. In addition, we assessed the behavioral responsiveness of mice to fluoxetine, desipramine, ketamine, and the Kv7 channel opener and anticonvulsant retigabine. HFD exposure led to glucose intolerance and neuroinflammation in male mice, with similar but non-significant trends in females. Neuroinflammation of males was associated with anxious-depressive-like behavior and defects in working memory, along with neural hyperexcitability and increased Ih currents of pyramidal cells in the PLC. The behavioral changes were largely resistant to chronic treatment with fluoxetine and desipramine, as well as ketamine. By contrast, retigabine (also known as ezogabine) normalized neural excitability and Ih currents recorded from slices of HFD-treated animals and significantly ameliorated most of the behavioral impairments, without effects in control diet exposed animals. Thus, treatment resistant depressive-like brain states that are associated with chronic neuroinflammation may involve hyperexcitability of pyramidal neurons and may be effectively treated by retigabine.

A trafficking-deficient KCNQ1 mutation, T587M, causes a severe phenotype of long QT syndrome by interfering with intracellular hERG transport.

BACKGROUND: KCNQ1-T587M is a C-terminal mutation correlated with severe phenotypes of long QT syndrome (LQTS). However, functional analysis of KCNQ1 channels with the T587M mutation showed a mild genotype in the form of haploinsufficiency in a heterologous expression system. This study sought to explore the molecular mechanism underlying the phenotype-genotype dissociation of LQTS patients carrying the KCNQ1-T587M mutation. METHODS: cDNAs for wild-type (WT) and KCNQ1 mutations (R259C and T587M) were transiently transfected into HEK293 cells stably expressing hERG (hERG-HEK), and whole-cell patch-clamp technique was performed to examine the effect of KCNQ1 mutations on IKr-like currents. In addition, fluorescence resonance energy transfer (FRET) was conducted to demonstrate the molecular interaction between KCNQ1 and hERG when co-expressed in HEK293 cells. RESULTS: KCNQ1-T587M mutation produced a significant (p<0.01) decrease in IKr-like tail current densities without affecting the gating kinetics, while KCNQ1-R259C mutation had no significant effect on the IKr-like tail current densities. Consistent with this result, FRET experiments demonstrated that both KCNQ1-WT and -R259C interacted with hERG in the cytosol and on the plasma membrane; however, the interaction between KCNQ1-T587M and hERG was observed only in the cytosol, and hERG proteins were seldom transported to the cell membrane, suggesting that the KCNQ1-T587M mutation impaired the trafficking of hERG to the cell membrane. CONCLUSIONS: The disruption of hERG trafficking caused by the KCNQ1-T587M mutation is likely the reason why some patients exhibit severe LQTS phenotypes.

K2P TASK-2 and KCNQ1-KCNE3 K+ channels are major players contributing to intestinal anion and fluid secretion.

KEY POINTS: K+ channels are important in intestinal epithelium as they ensure the ionic homeostasis and electrical potential of epithelial cells during anion and fluid secretion. Intestinal epithelium cAMP-activated anion secretion depends on the activity of the (also cAMP dependent) KCNQ1-KCNE3 K+ channel, but the secretory process survives after genetic inactivation of the K+ channel in the mouse. Here we use double mutant mice to investigate which alternative K+ channels come into action to compensate for the absence of KCNQ1-KCNE3 K+ channels. Our data establish that whilst Ca2+ -activated KCa 3.1 channels are not involved, K2P two-pore domain TASK-2 K+ channels are major players providing an alternative conductance to sustain the intestinal secretory process. Work with double mutant mice lacking both TASK-2 and KCNQ1-KCNE3 channels nevertheless points to yet-unidentified K+ channels that contribute to the robustness of the cAMP-activated anion secretion process. ABSTRACT: Anion and fluid secretion across the intestinal epithelium, a process altered in cystic fibrosis and secretory diarrhoea, is mediated by cAMP-activated CFTR Cl- channels and requires the simultaneous activity of basolateral K+ channels to maintain cellular ionic homeostasis and membrane potential. This function is fulfilled by the cAMP-activated K+ channel formed by the association of pore-forming KCNQ1 with its obligatory KCNE3 ß-subunit. Studies using mice show sizeable cAMP-activated intestinal anion secretion in the absence of either KCNQ1 or KCNE3 suggesting that an alternative K+ conductance must compensate for the loss of KCNQ1-KCNE3 activity. We used double mutant mouse and pharmacological approaches to identify such a conductance. Ca2+ -dependent anion secretion can also be supported by Ca2+ -dependent KCa 3.1 channels after independent CFTR activation, but cAMP-dependent anion secretion is not further decreased in the combined absence of KCa 3.1 and KCNQ1-KCNE3 K+ channel activity. We show that the K2P K+ channel TASK-2 is expressed in the epithelium of the small and large intestine. Tetrapentylammonium, a TASK-2 inhibitor, abolishes anion secretory current remaining in the absence of KCNQ1-KCNE3 activity. A double mutant mouse lacking both KCNQ1-KCNE3 and TASK-2 showed a much reduced cAMP-mediated anion secretion compared to that observed in the single KCNQ1-KCNE3 deficient mouse. We conclude that KCNQ1-KCNE3 and TASK-2 play major roles in the intestinal anion and fluid secretory phenotype. The persistence of an, admittedly reduced, secretory activity in the absence of these two conductances suggests that further additional K+ channel(s) as yet unidentified contribute to the robustness of the intestinal anion secretory process.

M-current inhibition rapidly induces a unique CK2-dependent plasticity of the axon initial segment.

Alterations in synaptic input, persisting for hours to days, elicit homeostatic plastic changes in the axon initial segment (AIS), which is pivotal for spike generation. Here, in hippocampal pyramidal neurons of both primary cultures and slices, we triggered a unique form of AIS plasticity by selectively targeting M-type K+ channels, which predominantly localize to the AIS and are essential for tuning neuronal excitability. While acute M-current inhibition via cholinergic activation or direct channel block made neurons more excitable, minutes to hours of sustained M-current depression resulted in a gradual reduction in intrinsic excitability. Dual soma-axon patch-clamp recordings combined with axonal Na+ imaging and immunocytochemistry revealed that these compensatory alterations were associated with a distal shift of the spike trigger zone and distal relocation of FGF14, Na+, and Kv7 channels but not ankyrin G. The concomitant distal redistribution of FGF14 together with Nav and Kv7 segments along the AIS suggests that these channels relocate as a structural and functional unit. These fast homeostatic changes were independent of l-type Ca2+ channel activity but were contingent on the crucial AIS protein, protein kinase CK2. Using compartmental simulations, we examined the effects of varying the AIS position relative to the soma and found that AIS distal relocation of both Nav and Kv7 channels elicited a decrease in neuronal excitability. Thus, alterations in M-channel activity rapidly trigger unique AIS plasticity to stabilize network excitability.

KCNE1 and KCNE3 modulate KCNQ1 channels by affecting different gating transitions.

KCNE ß-subunits assemble with and modulate the properties of voltage-gated K+ channels. In the heart, KCNE1 associates with the α-subunit KCNQ1 to generate the slowly activating, voltage-dependent potassium current (IKs) in the heart that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach, and kidney, KCNE3 coassembles with KCNQ1 to form K+ channels that are voltage-independent K+ channels in the physiological voltage range and important for controlling water and salt secretion and absorption. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by mutations or phosphatidylinositol 4,5-bisphosphate depletion, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 affects the S4 movement and only affects the gate in KCNQ1 if an intact S4-to-gate coupling is present. Further, we show that a triple mutation in the middle of the transmembrane (TM) segment of KCNE3 introduces KCNE1-like effects on the second S4 movement and the gate. In addition, we show that differences in two residues at the external end of the KCNE TM segments underlie differences in the effects of the different KCNEs on the first S4 movement and the voltage sensor-to-gate coupling.

Effects of 15 Hz square wave magnetic fields on the voltage-gated sodium and potassium channels in prefrontal cortex pyramidal neurons.

PURPOSE: Although magnetic fields have significant effects on neurons, little is known about the mechanisms behind their effects. The present study aimed to measure the effects of magnetic fields on ion channels in cortical pyramidal neurons. MATERIALS AND METHODS: Cortical pyramidal neurons of Kunming mice were isolated and then subjected to 15 Hz, 1 mT square wave (duty ratio 50%) magnetic fields stimulation. Sodium currents (INa), transient potassium currents (IA) and delayed rectifier potassium currents (IK) were recorded by whole-cell patch clamp method. RESULTS: We found that magnetic field exposure depressed channel current densities, and altered the activation kinetics of sodium and potassium channels. The inactivation properties of INa and IA were also altered. CONCLUSION: Magnetic field exposure alters ion channel function in neurons. It is likely that the structures of sodium and potassium channels were influenced by the applied field. Sialic acid, which is an important component of the channels, could be the molecule responsible for the reported results.

KCNE3 acts by promoting voltage sensor activation in KCNQ1.

KCNE ß-subunits assemble with and modulate the properties of voltage-gated K(+) channels. In the colon, stomach, and kidney, KCNE3 coassembles with the α-subunit KCNQ1 to form K(+) channels important for K(+) and Cl(-) secretion that appear to be voltage-independent. How KCNE3 subunits turn voltage-gated KCNQ1 channels into apparent voltage-independent KCNQ1/KCNE3 channels is not completely understood. Different mechanisms have been proposed to explain the effect of KCNE3 on KCNQ1 channels. Here, we use voltage clamp fluorometry to determine how KCNE3 affects the voltage sensor S4 and the gate of KCNQ1. We find that S4 moves in KCNQ1/KCNE3 channels, and that inward S4 movement closes the channel gate. However, KCNE3 shifts the voltage dependence of S4 movement to extreme hyperpolarized potentials, such that in the physiological voltage range, the channel is constitutively conducting. By separating S4 movement and gate opening, either by a mutation or PIP2 depletion, we show that KCNE3 directly affects the S4 movement in KCNQ1. Two negatively charged residues of KCNE3 (D54 and D55) are found essential for the effect of KCNE3 on KCNQ1 channels, mainly exerting their effects by an electrostatic interaction with R228 in S4. Our results suggest that KCNE3 primarily affects the voltage-sensing domain and only indirectly affects the gate.

Regulation of Voltage Gated K+ Channel KCNE1/KCNQ1 by the Janus Kinase JAK3.

BACKGROUND/AIMS: Janus kinase 3 (JAK3), a kinase mainly expressed in hematopoietic cells, has been shown to down-regulate the Na+/K+ ATPase and participate in the regulation of several ion channels and carriers. Channels expressed in thymus and regulating the abundance of T lymphocytes include the voltage gated K+ channel KCNE1/KCNQ1. The present study explored whether JAK3 contributes to the regulation of KCNE1/KCNQ1. METHODS: cRNA encoding KCNE1/KCNQ1 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild-type JAK3, constitutively active A568VJAK3, or inactive K851AJAK3. Voltage gated K+ channel activity was measured utilizing two electrode voltage clamp. RESULTS: KCNE1/KCNQ1 activity was significantly increased by wild-type JAK3 and A568VJAK3, but not by K851AJAK3. The difference between oocytes expressing KCNE1/KCNQ1 alone and oocytes expressing KCNE1/KCNQ1 with A568VJAK3 was virtually abrogated by JAK3 inhibitor WHI-P154 (22 µM) but not by inhibition of transcription with actinomycin D (50 nM). Inhibition of KCNE1/KCNQ1 protein insertion into the cell membrane by brefeldin A (5 µM) resulted in a decline of the voltage gated current, which was similar in the absence and presence of A568VJAK3, suggesting that A568VJAK3 did not accelerate KCNE1/KCNQ1 protein retrieval from the cell membrane. CONCLUSION: JAK3 contributes to the regulation of membrane KCNE1/KCNQ1 activity, an effect sensitive to JAK3 inhibitor WHI-P154.

A delayed chronic pain like condition with decreased Kv channel activity in a rat model of Gulf War Illness pain syndrome.

Following their return from deployment, Gulf War (GW) veterans reported widespread joint and muscle pain at rates that far exceeded those of soldiers returning from other conflicts. It is widely believed that exposure to insecticides, repellants and nerve gas prophylactics contributed to the symptoms of Gulf War Illness (GWI), but an animal model of GW pain has been elusive. In our previous work, we observed that 4-8 weeks exposure to pyridostigmine bromide (PB), permethrin and chlorpyrifos could produce persistent alterations in the physiology of Nav1.9 and Kv7 expressed in deep tissue nociceptors of the dorsal root ganglion. However, behavioral assessments from these same rats were not consistent with a delayed pain syndrome similar to that of GWI pain. In the present studies, we intensified the exposure to anticholinesterases PB and chlorpyrifos while retaining the same dosages. Animals receiving the intensified protocol for 30 days exhibited significant increases in resting for about 8 weeks after exposure. Thereafter, all measures were comparable to controls. Animals treated with intensified anticholinesterases for 60 days exhibited increased resting and reduced movement 12 weeks post-exposure. In whole cell patch studies, muscle and vascular nociceptor KDR and Kv7 ion channels exhibited increased amplitude relative to controls (e.g., normalized current and/or peak conductance) at 8 weeks post-exposures; however, at 12 weeks post-exposure, the amplitude of these currents was significantly decreased in muscle nociceptors. In current clamp studies, muscle nociceptors also manifested increased action potential duration, afterhyperpolarization and increased discharge to muscarinic agonists 12 weeks post-exposure. The decline in activity of muscle nociceptor KDR and Kv7 channel proteins was consistent with increased nociceptor excitability and a delayed myalgia in rats exposed to GW chemicals.

Partial Loss of Genomic Imprinting Reveals Important Roles for Kcnq1 and Peg10 Imprinted Domains in Placental Development.

Mutations in imprinted genes or their imprint control regions (ICRs) produce changes in imprinted gene expression and distinct abnormalities in placental structure, indicating the importance of genomic imprinting to placental development. We have recently shown that a very broad spectrum of placental abnormalities associated with altered imprinted gene expression occurs in the absence of the oocyte-derived DNMT1o cytosine methyltransferase, which normally maintains parent-specific imprinted methylation during preimplantation. The absence of DNMT1o partially reduces inherited imprinted methylation while retaining the genetic integrity of imprinted genes and their ICRs. Using this novel system, we undertook a broad and inclusive approach to identifying key ICRs involved in placental development by correlating loss of imprinted DNA methylation with abnormal placental phenotypes in a mid-gestation window (E12.5-E15.5). To these ends we measured DNA CpG methylation at 15 imprinted gametic differentially methylated domains (gDMDs) that overlap known ICRs using EpiTYPER-mass array technology, and linked these epigenetic measurements to histomorphological defects. Methylation of some imprinted gDMDs, most notably Dlk1, was nearly normal in mid-gestation DNMT1o-deficient placentas, consistent with the notion that cells having lost methylation on these DMDs do not contribute significantly to placental development. Most imprinted gDMDs however showed a wide range of methylation loss among DNMT1o-deficient placentas. Two striking associations were observed. First, loss of DNA methylation at the Peg10 imprinted gDMD associated with decreased embryonic viability and decreased labyrinthine volume. Second, loss of methylation at the Kcnq1 imprinted gDMD was strongly associated with trophoblast giant cell (TGC) expansion. We conclude that the Peg10 and Kcnq1 ICRs are key regulators of mid-gestation placental function.

An Epithelial Ca2+-Sensor Protein is an Alternative to Calmodulin to Compose Functional KCNQ1 Channels.

BACKGROUND/AIMS: KCNQ channels transport K+ ions and participate in various cellular functions. The channels directly assemble with auxiliary proteins such as a ubiquitous Ca2+- sensor protein, calmodulin (CaM), to configure the physiological properties in a tissue-specific manner. Although many CaM-like Ca2+-sensor proteins have been identified in eukaryotes, how KCNQ channels selectively interact with CaM and how the homologues modulate the functionality of the channels remain unclear. METHODS: We developed protocols to evaluate the interaction between the green fluorescent protein-tagged C-terminus of KCNQ1 (KCNQ1cL) and Ca2+-sensors by detecting its fluorescence in size exclusion chromatography and electrophoresed gels. The effects of Ca2+-sensor proteins on KCNQ1 activity was measured by two electrode voltage clamp technique of Xenopus oocytes. RESULTS: When co-expressed CaM and KCNQ1cL, they assemble in a 4:4 stoichiometry, forming a hetero-octamer. Among nine CaM homologues tested, Calml3 was found to form a hetero-octamer with KCNQ1cL and to associate with the full-length KCNQ1 in a competitive manner with CaM. When co-expressed in oocytes, Calml3 rendered KCNQ1 channels resistant to the voltage-dependent depletion of phosphatidylinositol 4,5-bisphosphate by voltage-sensitive phosphatase. CONCLUSION: Since Calml3 is closely related to CaM and is prominently expressed in epithelial cells, Calml3 may be a constituent of epithelial KCNQ1 channels and underscores the molecular diversity of endogenous KCNQ1 channels.

The KCNQ1 channel - remarkable flexibility in gating allows for functional versatility.

The KCNQ1 channel (also called Kv7.1 or KvLQT1) belongs to the superfamily of voltage-gated K(+) (Kv) channels. KCNQ1 shares several general features with other Kv channels but also displays a fascinating flexibility in terms of the mechanism of channel gating, which allows KCNQ1 to play different physiological roles in different tissues. This flexibility allows KCNQ1 channels to function as voltage-independent channels in epithelial tissues, whereas KCNQ1 function as voltage-activated channels with very slow kinetics in cardiac tissues. This flexibility is in part provided by the association of KCNQ1 with different accessory KCNE ß-subunits and different modulators, but also seems like an integral part of KCNQ1 itself. The aim of this review is to describe the main mechanisms underlying KCNQ1 flexibility.

Kv7 channels critically determine coronary artery reactivity: left-right differences and down-regulation by hyperglycaemia.

AIMS: Voltage-gated potassium channels encoded by KCNQ genes (Kv7 channels) are emerging as important regulators of vascular tone. In this study, we analysed the contribution of Kv7 channels to the vasodilation induced by hypoxia and the cyclic AMP pathway in the coronary circulation. We also assessed their regional distribution and possible impairment by diabetes. METHODS AND RESULTS: We examined the effects of Kv7 channel modulators on K+ currents and vascular reactivity in rat left and right coronary arteries (LCAs and RCAs, respectively). Currents from LCA were more sensitive to Kv7 channel inhibitors (XE991, linopirdine) and activators (flupirtine, retigabine) than those from RCA. Accordingly, LCAs were more sensitive than RCAs to the relaxation induced by Kv7 channel enhancers. Likewise, relaxation induced by the adenylyl cyclase activator forskolin and hypoxia, which were mediated through Kv7 channel activation, were greater in LCA than in RCA. KCNQ1 and KCNQ5 expression was markedly higher in LCA than in RCA. After incubation with high glucose (HG, 30 mmol/L), myocytes from LCA, but not from RCA, were more depolarized and showed reduced Kv7 currents. In HG-incubated LCA, the effects of Kv7 channel modulators and forskolin were diminished, and the expression of KCNQ1 and KCNQ5 was reduced. Finally, vascular responses induced by Kv7 channel modulators were impaired in LCA, but not in RCA, from type 1 diabetic rats. CONCLUSION: Our results reveal that the high expression and function of Kv7 channels in the LCA and their down-regulation by diabetes critically determine the sensitivity to key regulators of coronary tone.

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.

Cellular mechanisms underlying the increased disease severity seen for patients with long QT syndrome caused by compound mutations in KCNQ1.

The KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene encodes the Kv7.1 potassium channel which forms a complex with KCNE1 (potassium voltage-gated channel Isk-related family member 1) in the human heart to produce the repolarizing IKs (slow delayed rectifier potassium current). Mutations in KCNQ1 can perturb IKs function and cause LQT1 (long QT syndrome type 1). In LQT1, compound mutations are relatively common and are associated with increased disease severity. LQT1 compound mutations have been shown to increase channel dysfunction, but whether other disease mechanisms, such as defective channel trafficking, contribute to the increase in arrhythmic risk has not been determined. Using an imaging-based assay we investigated the effects of four compound heterozygous mutations (V310I/R594Q, A341V/P127T, T391I/Q530X and A525T/R518X), one homozygous mutation (W248F) and one novel compound heterozygous mutation (A178T/K422fs39X) (where fs denotes frameshift) on channel trafficking. By analysing the effects in the equivalent of a homozygous, heterozygous and compound heterozygous condition, we identify three different types of behaviour. A341V/P127T and W248F/W248F had no effect, whereas V310I/R594Q had a moderate, but not compound, effect on channel trafficking. In contrast, T391I/Q530X, A525T/R518X and A178T/K422fs39X severely disrupted channel trafficking when expressed in compound form. In conclusion, we have characterized the disease mechanisms for six LQT1 compound mutations and report that, for four of these, defective channel trafficking underlies the severe clinical phenotype.

Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain.

A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K(+) current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure-function relation of KCNQ1 channel and treatments of cardiac channelopathies.