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.

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.

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.

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.

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.

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.

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.

Interactions between hERG and KCNQ1 α-subunits are mediated by their COOH termini and modulated by cAMP.

KCNQ1 and hERG encode the voltage-gated potassium channel α-subunits of the cardiac repolarizing currents I(Ks) and I(Kr), respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs), and loss-of-function mutations in these channels manifest clinically as long QT syndrome, characterized by the prolongation of the QT interval, polymorphic ventricular tachycardia, and sudden cardiac death. Previous cellular electrophysiology experiments in transgenic rabbit cardiomyocytes and heterologous cell lines demonstrated functional downregulation of complementary repolarizing currents. Biochemical assays indicated direct, protein-protein interactions between KCNQ1 and hERG may underlie the interplay between I(Ks) and I(Kr). Our objective was to investigate hERG-KCNQ1 interactions in the intact cellular environment primarily through acceptor photobleach FRET (apFRET) experiments. We quantitatively assessed the extent of interactions based on fluorophore location and the potential regulation of interactions by physiologically relevant signals. apFRET experiments established specific hERG-KCNQ1 associations in both heterologous and primary cardiomyocytes. The largest FRET efficiency (E(f); 12.0 ± 5.2%) was seen between ion channels with GFP variants fused to the COOH termini. Acute treatment with forskolin + IBMX or a membrane-permeable cAMP analog significantly and specifically reduced the extent of hERG-KCNQ1 interactions (by 41 and 38%, respectively). Our results demonstrate direct interactions between KCNQ1 and hERG occur in both intact heterologous cells and primary cardiomyocytes and are mediated by their COOH termini. Furthermore, this interplay between channel proteins is regulated by intracellular cAMP.

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.

Readthrough of long-QT syndrome type 1 nonsense mutations rescues function but alters the biophysical properties of the channel.

The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause LQT1 (long-QT syndrome type 1) and result in a complete loss of I(Ks) channel function. In the present study we attempted to rescue the function of these mutants, in HEK (human embryonic kidney)-293 cells, by promoting readthrough of their PTCs (premature termination codons) using the pharmacological agents G-418, gentamicin and PTC124. Gentamicin and G-418 acted to promote full-length channel protein expression from R518X at 100 µM and from Q530X at 1 mM. In contrast, PTC124 did not, at any dose tested, induce readthrough of either mutant. G-418 (1 mM) treatment also acted to significantly (P<0.05) increase current density and peak-tail current density, at +80 mV for R518X, but not Q530X, to 58±11% and 82±17% of the wild-type level respectively. However, the biophysical properties of the currents produced from R518X, while similar, were not identical with wild-type as the voltage-dependence of activation was significantly (P<0.05) shifted by +25 mV. Overall, these findings indicate that although functional rescue of LQT1 nonsense mutations is possible, it is dependent on the degree of readthrough achieved and the effect on channel function of the amino acid substituted for the PTC. Such considerations will determine the success of future therapies.

KCNE1 alters the voltage sensor movements necessary to open the KCNQ1 channel gate.

The delayed rectifier I(Ks) potassium channel, formed by coassembly of α- (KCNQ1) and ß- (KCNE1) subunits, is essential for cardiac function. Although KCNE1 is necessary to reproduce the functional properties of the native I(Ks) channel, the mechanism(s) through which KCNE1 modulates KCNQ1 is unknown. Here we report measurements of voltage sensor movements in KCNQ1 and KCNQ1/KCNE1 channels using voltage clamp fluorometry. KCNQ1 channels exhibit indistinguishable voltage dependence of fluorescence and current signals, suggesting a one-to-one relationship between voltage sensor movement and channel opening. KCNE1 coexpression dramatically separates the voltage dependence of KCNQ1/KCNE1 current and fluorescence, suggesting an imposed requirement for movements of multiple voltage sensors before KCNQ1/KCNE1 channel opening. This work provides insight into the mechanism by which KCNE1 modulates the I(Ks) channel and presents a mechanism for distinct ß-subunit regulation of ion channel proteins.

High-risk long QT syndrome mutations in the Kv7.1 (KCNQ1) pore disrupt the molecular basis for rapid K(+) permeation.

Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the K(+) channel (Kv7.1) that underlies the slowly activating delayed rectifier K(+) current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss of function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confers a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated nonfunctional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamics simulations of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K(+)-K(+) repulsive forces required for rapid K(+) permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K(+) channel selectivity filter.

Pro-arrhythmogenic effects of the S140G KCNQ1 mutation in human atrial fibrillation - insights from modelling.

Functional analysis has shown that the missense gain-in-function KCNQ1 S140G mutation associated with familial atrial fibrillation produces an increase of the slow delayed rectifier potassium current (I(Ks)). Through computer modelling, this study investigated mechanisms by which the KCNQ1 S140G mutation promotes and perpetuates atrial fibrillation. In simulations, Courtemanche et al.'s model of human atrial cell action potentials (APs) was modified to incorporate experimental data on changes of I(Ks) induced by the KCNQ1 S140G mutation. The cell models for wild type (WT) and mutant type (MT) I(Ks) were incorporated into homogeneous multicellular 2D and 3D tissue models. Effects of the mutation were quantified on AP profile, AP duration (APD) restitution, effective refractory period (ERP) restitution, and conduction velocity (CV) restitution.Temporal and spatial vulnerabilities of atrial tissue to genesis of re-entry were computed. Dynamic behaviours of re-entrant excitation waves (lifespan (LS), tip meandering patterns and dominant frequency) in 2D and 3D models were characterised. It was shown that the KCNQ1 S140G mutation abbreviated atrial APD and ERP and flattened APD and ERP restitution curves. It reduced atrial CV at low excitation rates, but increased it at high excitation rates that facilitated the conduction of high rate atrial excitation waves. Although it increased slightly tissue temporal vulnerability for initiating re-entry, it reduced markedly the minimal substrate size necessary for sustaining re-entry (increasing the tissue spatial vulnerability). In the 2D and 3D models, the mutation also stabilized and accelerated re-entrant excitation waves, leading to rapid and sustained re-entry. In the 3D model, scroll waves under the mutation condition MT conditions also degenerated into persistent and erratic wavelets, leading to fibrillation. In conclusion, increased I(Ks) due to the KCNQ1 S140G mutation increases atrial susceptibility to arrhythmia due to increased tissue vulnerability, shortened ERP and altered atrial conduction velocity, which, in combination, facilitate initiation and maintenance of re-entrant excitation waves.

Overlapping cardiac phenotype associated with a familial mutation in the voltage sensor of the KCNQ1 channel.

BACKGROUND: Cardiac action potential repolarisation is determined by K(+) currents including I(Ks). I(Ks) channels are heteromeric channels composed of KCNQ1 and KCNE E-subunits. Mutations in KCNQ1 are associated with sinus bradycardia, familial atrial fibrillation (fAF) and/or short QT syndrome as a result of gain-of-function, and long QT syndrome (LQTS) due to loss-of-function in the ventricles. Here, we report that the missense mutation R231C located in S4 voltage sensor domain is associated with a combined clinical phenotype of sinus bradycardia, fAF and LQTS. We aim to understand the molecular basis of the complex clinical phenotype. METHODS: We expressed and functionally analyzed the respective channels kinetics in Xenopus laevis oocytes. The molecular nature of the residue R231 was studied by homology modeling and molecular dynamics simulation. RESULTS: As a result, the mutation reduced voltage sensitivity of channels, possibly due to neutralization of the positive charge of the arginine side chain substituted by cysteine. Modeling suggested that the charge carrying side chain of R231 is positioned suitably to transfer transmembrane voltages into conformational energy. Further, the mutation altered the functional interactions with KCNE subunits. CONCLUSION: The mutation acted in a E-subunit dependent manner, suggesting I(Ks) function altered by the presence of different KCNE subunits in sinus node, atria and ventricles as the molecular basis of sinus bradycardia, fAF and LQTS in mutation carriers.

Location of KCNE1 relative to KCNQ1 in the I(KS) potassium channel by disulfide cross-linking of substituted cysteines.

The cardiac-delayed rectifier K(+) current (I(KS)) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic I(KS) voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cys-flanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.

Biophysical properties of slow potassium channels in human embryonic stem cell derived cardiomyocytes implicate subunit stoichiometry.

Human embryonic stem cells (hESCs) are an important cellular model for studying ion channel function in the context of a human cardiac cell and will provide a wealth of information about both heritable arrhythmias and acquired electrophysiological disorders. However, detailed electrophysiological characterization of the important cardiac ion channels has been so far overlooked. Because mutations in the gene for the I(Ks) α subunit, KCNQ1, constitute the majority of long QT syndrome (LQT-1) cases, we have carried out a detailed biophysical analysis of this channel expressed in hESCs to establish baseline I(Ks) channel biophysical properties in cardiac myocytes derived from hESCs (hESC-CMs). I(Ks) channels are heteromultimeric proteins consisting of four identical α-subunits (KCNQ1) assembled with auxiliary ß-subunits (KCNE1). We found that the half-maximal I(Ks) activation voltage in hESC-CMs and in myocytes derived from human induced pluripotent stems cells (hiPSC-CMs) falls between that of KCNQ1 channels expressed alone and with full complement of KCNE1, the major KCNE subunit expressed in hESC-CMs as shown by qPCR analysis. Overexpression of KCNE1 by transfection of hESC-CMs markedly shifted and slowed native I(Ks) activation implying assembly of additional KCNE1 subunits with endogenous channels. Our results in hESC-CMs, which indicate an I(Ks) subunit stoichiometry that can be altered by variable KCNE1 expression, suggest the possibility for variable I(Ks) function in the developing heart, in different tissues in the heart, and in disease. This establishes a new baseline for I(Ks) channel properties in myocytes derived from pluripotent stem cells and will guide future studies in patient-specific hiPSCs.

Sexual dimorphism and oestrogen regulation of KCNE3 expression modulates the functional properties of KCNQ1 K⁺ channels.

The KCNQ1 potassium channel associates with various KCNE ancillary subunits that drastically affect channel gating and pharmacology. Co-assembly with KCNE3 produces a current with nearly instantaneous activation, some time-dependent activation at very positive potentials, a linear current-voltage relationship and a 10-fold higher sensitivity to chromanol 293B. KCNQ1:KCNE3 channels are expressed in colonic crypts and mediate basolateral K(+) recycling required for Cl(-) secretion. We have previously reported the female-specific anti-secretory effects of oestrogen via KCNQ1:KCNE3 channel inhibition in colonic crypts. This study was designed to determine whether sex and oestrogen regulate the expression and function of KCNQ1 and KCNE3 in rat distal colon. Colonic crypts were isolated from Sprague-Dawley rats and used for whole-cell patch-clamp and to extract total RNA and protein. Sheets of epithelium were used for short-circuit current recordings. KCNE1 and KCNE3 mRNA and protein abundance were significantly higher in male than female crypts. No expression of KCNE2 was found and no difference was observed in KCNQ1 expression between male and female (at oestrus) colonic crypts. Male crypts showed a 2.2-fold higher level of association of KCNQ1 and KCNE3 compared to female cells. In female colonic crypts, KCNQ1 and KCNE3 protein expression fluctuated throughout the oestrous cycle and 17ß-oestradiol (E2 10 nM) produced a rapid (<15 min) dissociation of KCNQ1 and KCNE3 in female crypts only. Whole-cell K(+) currents showed a linear current-voltage relationship in male crypts, while K(+) currents in colonic crypts isolated from females displayed voltage-dependent outward rectification. Currents in isolated male crypts and epithelial sheets were 10-fold more sensitive to specific KCNQ1 inhibitors, such as chromanol 293B and HMR-1556, than in female. The effect of E2 on K(+) currents mediated by KCNQ1 with or without different ß-subunits was assayed from current-voltage relations elicited in CHO cells transfected with KCNQ1 and KCNE3 or KCNE1 cDNA. E2 (100 nM) reduced the currents mediated by the KCNQ1:KCNE3 potassium channel and had no effect on currents via KCNQ1:KCNE1 or KCNQ1 alone. Currents mediated by the complex formed by KCNQ1 and the mutant KCNE3-S82A ß-subunit (mutation of the site for PKCδ-promoted phosphorylation and modulation of the activity of KCNE3) showed rapid run-down and insensitivity to E2. Together, these data suggest that oestrogen regulates the expression of the KCNE1 and KCNE3 and with it the gating and pharmacological properties of the K(+) conductance required for Cl(-) secretion. The decreased association of the KCNQ1:KCNE3 channel complex promoted by oestrogen exposure underlies the molecular mechanism for the sexual dimorphism and oestrous cycle dependence of the anti-secretory actions of oestrogen in the intestine.

Differential effects of Kv7 (M-) channels on synaptic integration in distinct subcellular compartments of rat hippocampal pyramidal neurons.

The K(V)7/M-current is an important determinant of neuronal excitability and plays a critical role in modulating action potential firing. In this study, using a combination of electrophysiology and computational modelling, we show that these channels selectively influence peri-somatic but not dendritic post-synaptic excitatory synaptic potential (EPSP) integration in CA1 pyramidal cells. K(V)7/M-channels are highly concentrated in axons. However, the competing peptide, ankyrin G binding peptide (ABP) that disrupts axonal K(V)7/M-channel function, had little effect on somatic EPSP integration, suggesting that this effect was due to local somatic channels only. This interpretation was confirmed using computer simulations. Further, in accordance with the biophysical properties of the K(V)7/M-current, the effect of somatic K(V)7/M-channels on synaptic potential summation was dependent upon the neuronal membrane potential. Somatic K(V)7/M-channels thus affect EPSP-spike coupling by altering EPSP integration. Interestingly, disruption of axonal channels enhanced EPSP-spike coupling by lowering the action potential threshold. Hence, somatic and axonal K(V)7/M-channels influence EPSP-spike coupling via different mechanisms. This may be important for their relative contributions to physiological processes such as synaptic plasticity as well as patho-physiological conditions such as epilepsy.

Direct observation of individual KCNQ1 potassium channels reveals their distinctive diffusive behavior.

We have directly observed the trafficking and fusion of ion channel containing vesicles and monitored the release of individual ion channels at the plasma membrane of live mammalian cells using total internal reflection fluorescence microscopy. Proteins were fused in-frame with green or red fluorescent proteins and expressed at low level in HL-1 and HEK293 cells. Dual color imaging revealed that vesicle trafficking involved motorized movement along microtubules followed by stalling, fusion, and subsequent release of individual ion channels at the plasma membrane. We found that KCNQ1-KCNE1 complexes were released in batches of about 5 molecules per vesicle. To elucidate the properties of ion channel complexes at the cell membrane we tracked the movement of individual molecules and compared the diffusive behavior of two types of potassium channel complex (KCNQ1-KCNE1 and Kir6.2-SUR2A) to that of a G-protein coupled receptor, the A1 adenosine receptor. Plots of mean squared displacement against time intervals showed that mobility depended on channel type, cell type, and temperature. Analysis of the mobility of wild type KCNQ1-KCNE1 complexes showed the existence of a significant immobile subpopulation and also a significant number of molecules that demonstrated periodic stalling of diffusive movements. This behavior was enhanced in cells treated with jasplakinolide and was abrogated in a C-terminal truncated form (KCNQ1(R518X)-KCNE1) of the protein. This mutant has been identified in patients with the long QT syndrome. We propose that KCNQ1-KCNE1 complexes interact intermittently with the actin cytoskeleton via the C-terminal region and this interaction may have a functional role.