Positive Shift In Voltage Dependence Research Articles

A positively charged residue at the Kv1.1 T1 interface is critical for voltage-dependent activation and gating kinetics.

Within the tetramerization domain (T1) of most voltage-gated potassium channels (Kv) are highly conserved charged residues that line the T1-T1 interface. We investigated the Kv1.1 residue R86 located at the narrowest region of the T1 interface. A Kv1.1 R86Q mutation was reported in a child diagnosed with lower limb dyskinesia [1]. The child did not present with episodic ataxia 1 (EA1) symptoms typically associated with Kv1.1 loss-of-function mutations. We characterized the electrophysiological outcome of the R86Q substitution by expressing Kv1.1 in Xenopus laevis oocytes. Mutated α-subunits were able to form functional channels that pass delayed rectifier currents. Oocytes that expressed only mutated α-subunits produced a significant reduction in Kv1.1 current and showed a positive shift in voltage-dependence of activation. In addition, there was substantially slower activation and faster deactivation implying a reduction in the time the channel is in its open state. Oocytes co-injected with both mutated and wild-type cRNA in equal amounts, to mimic the heterozygous condition of the disease, showed a decrease in current amplitude at -10mV and faster deactivation kinetics when compared to the wild-type channel. These findings indicate that T1 plays a role in Kv1.1's voltage-dependent activation and in its kinetics of activation and deactivation.

Recurrent Pregnancy Loss and Concealed Long‐QT Syndrome

BackgroundRecurrent pregnancy loss affects 1% to 2% of couples attempting childbirth. A large fraction of all cases remains idiopathic, which warrants research into monogenic causes of this distressing disorder.Methods and ResultsWe investigated a nonconsanguineous Estonian family who had experienced 5 live births, intersected by 3 early pregnancy losses, and 6 fetal deaths, 3 of which occurred during the second trimester. No fetal malformations were described at the autopsies performed in 3 of 6 cases of fetal death. Parental and fetal chromosomal abnormalities (including submicroscopic) and maternal risk factors were excluded. Material for genetic testing was available from 4 miscarried cases (gestational weeks 11, 14, 17, and 18). Exome sequencing in 3 pregnancy losses and the mother identified no rare variants explicitly shared by the miscarried conceptuses. However, the mother and 2 pregnancy losses carried a heterozygous nonsynonymous variant, resulting in p.Val173Asp (rs199472695) in the ion channel gene KCNQ1. It is expressed not only in heart, where mutations cause type 1 long‐QT syndrome, but also in other tissues, including uterus. The p.Val173Asp variant has been previously identified in a patient with type 1 long‐QT syndrome, but not reported in the Genome Aggregation Database. With heterologous expression in CHO cells, our in vitro electrophysiologic studies indicated that the mutant slowly activating voltage‐gated K+ channel (I Ks) is dysfunctional. It showed reduced total activating and deactivating currents (P<0.01), with dramatically positive shift of voltage dependence of activation by ≈10 mV (P<0.05).ConclusionsThe current study uncovered concealed maternal type 1 long‐QT syndrome as a potential novel cause behind recurrent fetal loss.

Kinetic Membrane Model of Outer Hair Cells

The effectiveness of outer hair cells (OHCs) in amplifying the motion of the organ of Corti, and thereby contributing to the sensitivity of mammalian hearing, depends on the mechanical power output of these cells. Electromechanical coupling in OHCs, which enables these cells to convert electrical energy into mechanical energy, has been analyzed in detail using isolated cells using primarily static membrane models. The mechanical output of OHCs was previously evaluated by developing a kinetic theory based on a simplified one-dimensional model for OHCs. Here, a kinetic description of OHCs is extended by using the membrane model, which was used for analyzing in vitro experiments. This theory predicts, for systems without inertial load, that elastic load enhances positive shift of voltage dependence of the membrane capacitance because of turgor pressure. The effect of turgor pressure increases with increasing elastic load. For systems with inertia, the magnitude of mechanical power output could be ∼5% higher than the value predicted by the one-dimensional model at the optimal turgor pressure.

A sustained increase in the intracellular Ca2+ concentration induces proteolytic cleavage of EAG2 channel

Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca2+ concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca2+ ionophore A23187 for 1h reduces the full-length protein by ∼80% with a concomitant appearance of 30–35-kDa peptides. Similarly, a treatment with the Ca2+-ATPase inhibitor thapsigargin for 3h removes 30–35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl2 leads to the generation of fragments with similar sizes. This Ca2+-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca2+ concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca2+-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.

Slow Delayed Rectifier Potassium Current Blockade Contributes Importantly to Drug-Induced Long QT Syndrome

Background— Drug-induced long QT syndrome is generally ascribed to inhibition of the cardiac rapid delayed rectifier potassium current ( I Kr ). Effects on the slow delayed rectifier potassium current ( I Ks ) are less recognized. Triggered by a patient who carried the K422T mutation in KCNQ1 (encoding the α-subunit of the I Ks channel), who presented with excessive QT prolongation and high serum levels of norfluoxetine, we investigated the effects of fluoxetine and its metabolite norfluoxetine on I Ks . Methods and Results— ECG data from mutation carriers and noncarriers revealed that the K422T mutation per se had mild clinical effects. Patch clamp studies, performed on HEK293 cells, showed that heterozygously expressed K422T KCNQ1/KCNE1 channels had a positive shift in voltage dependence of activation and an increase in deactivation rate. Fluoxetine and its metabolite norfluoxetine both inhibited KCNQ1/KCNE1 current, with norfluoxetine being the most potent. Moreover, norfluoxetine increased activation and deactivation rates. Computer simulations of the effects of norfluoxetine on I Ks and I Kr demonstrated significant action potential prolongation, to which I Ks block contributed importantly. Although the effects of the mutation per se were small, additional I Ks blockade by norfluoxetine resulted in more prominent QTc prolongation in mutation carriers than in noncarriers, demonstrating synergistic effects of innate and drug-induced I Ks blockade on QTc prolongation. Conclusions— I Ks blockade contributes importantly to drug-induced long QT syndrome, especially when repolarization reserve is reduced. Drug safety tests might have to include screening for I Ks blockade.

KCNE2 modulation of Kv4.3 current and its potential role in fatal rhythm disorders

The transient outward current I(to) is of critical importance in regulating myocardial electrical properties during the very early phase of the action potential. The auxiliary beta subunit KCNE2 recently was shown to modulate I(to). The purpose of this study was to examine the contributions of KCNE2 and its two published variants (M54T, I57T) to I(to). The functional interaction between Kv4.3 (alpha subunit of human I(to)) and wild-type (WT), M54T, and I57T KCNE2, expressed in a heterologous cell line, was studied using patch-clamp techniques. Compared to expression of Kv4.3 alone, co-expression of WT KCNE2 significantly reduced peak current density, slowed the rate of inactivation, and caused a positive shift of voltage dependence of steady-state inactivation curve. These modifications rendered Kv4.3 channels more similar to native cardiac I(to). Both M54T and I57T variants significantly increased I(to) current density and slowed the inactivation rate compared with WT KCNE2. Moreover, both variants accelerated the recovery from inactivation. The study results suggest that KCNE2 plays a critical role in the normal function of the native I(to) channel complex in human heart and that M54T and I57T variants lead to a gain of function of I(to), which may contribute to generating potential arrhythmogeneity and pathogenesis for inherited fatal rhythm disorders.

Gain-of-function mutation of Na v1.5 in atrial fibrillation enhances cellular excitability and lowers the threshold for action potential firing

Genetic mutations of the cardiac sodium channel (SCN5A) specific only to the phenotype of atrial fibrillation have recently been described. However, data on the biophysical properties of SCN5A variants associated with atrial fibrillation are scarce. In a mother and son with lone atrial fibrillation, we identified a novel SCN5A coding variant, K1493R, which altered a highly conserved residue in the DIII–IV linker and was located six amino acids downstream from the fast inactivation motif of sodium channels. Biophysical studies of K1493R in tsA201 cells demonstrated a significant positive shift in voltage-dependence of inactivation and a large ramp current near resting membrane potential, indicating a gain-of-function. Enhanced cellular excitability was observed in transfected HL-1 atrial cardiomyocytes, including spontaneous action potential depolarizations and a lower threshold for action potential firing. These novel biophysical observations provide molecular evidence linking cellular “hyperexcitability” as a mechanism inducing vulnerability to this common arrhythmia.

Wild-Type and Mutant HCN Channels in a Tandem Biological-Electronic Cardiac Pacemaker

Biological pacemakers (BPM) implanted in canine left bundle branch function competitively with electronic pacemakers (EPM). We hypothesized that BPM engineered with the use of mE324A mutant murine HCN2 (mHCN2) genes would improve function over mHCN2 and that BPM/EPM tandems confer advantage over either approach alone. In cultured neonatal rat myocytes, activation midpoint was -46.9 mV in mE324A versus -66.1 mV in mHCN2 (P < 0.05). mE324A manifested a positive shift of voltage dependence of gating kinetics of activation and deactivation compared with mHCN2 (P < 0.05) in myocytes as well as Xenopus oocytes. In intact dogs in complete atrioventricular block, saline (control), mHCN2, or mE324A virus was injected into left bundle branch, and EPM were implanted (VVI 45 bpm). Twenty-four-hour ECGs were monitored for 14 days. With EPM discontinued, there was no difference in duration of overdrive suppression among groups. However, basal heart rates in controls were less than those in mHCN2, which did not differ from those in E324A (45 versus 57 versus 53 bpm; P < 0.05). When spontaneous rate fell below 45 bpm, EPM intervened at that rate, triggering 83% of beats in control, contrasting (P < 0.05) with 26% (mHCN2) and 36% (mE324A). On day 14, epinephrine (1 microg/kg per minute IV) induced a 50% heart rate increase in all mE324A, one third of mHCN2, and one fifth of control (P < 0.05 mE324A versus control or mHCN2). mE324A induces faster, more positive pacemaker current activation than mHCN2 and stable, catecholamine-sensitive rhythms in situ that compete with EPM comparably but more catecholamine responsively than mHCN2. BPM/EPM tandems function reliably, reduce the number of EPM beats, and confer sympathetic responsiveness to the tandem.

Characterization of N‐glycosylation consensus sequences in the Kv3.1 channel

N-Glycosylation is a cotranslational and post-translational process of proteins that may influence protein folding, maturation, stability, trafficking, and consequently cell surface expression of functional channels. Here we have characterized two consensus N-glycosylation sequences of a voltage-gated K+ channel (Kv3.1). Glycosylation of Kv3.1 protein from rat brain and infected Sf9 cells was demonstrated by an electrophoretic mobility shift assay. Digestion of total brain membranes with peptide N glycosidase F (PNGase F) produced a much faster-migrating Kv3.1 immunoband than that of undigested brain membranes. To demonstrate N-glycosylation of wild-type Kv3.1 in Sf9 cells, cells were treated with tunicamycin. Also, partially purified proteins were digested with either PNGase F or endoglycosidase H. Attachment of simple-type oligosaccharides at positions 220 and 229 was directly shown by single (N229Q and N220Q) and double (N220Q/N229Q) Kv3.1 mutants. Functional measurements and membrane fractionation of infected Sf9 cells showed that unglycosylated Kv3.1s were transported to the plasma membrane. Unitary conductance of N220Q/N229Q was similar to that of the wild-type Kv3.1. However, whole cell currents of N220Q/N229Q channels had slower activation rates, and a slight positive shift in voltage dependence compared to wild-type Kv3.1. The voltage dependence of channel activation for N229Q and N220Q was much like that for N220Q/N229Q. These results demonstrate that the S1-S2 linker is topologically extracellular, and that N-glycosylation influences the opening of the voltage-dependent gate of Kv3.1. We suggest that occupancy of the sites is critical for folding and maturation of the functional Kv3.1 at the cell surface.

Regulation of the Slow Vacuolar Channel by Luminal Potassium: Role of Surface Charge

Voltage-dependent activation of slow vacuolar (SV) channels has been studied on isolated patches from red beet (Beta vulgaris L.) vacuoles. Isoosmotic variation of vacuolar K(+) from 10 to 400 mM in Ca(2+)-free solutions at the vacuolar side shifted the SV channel activation threshold to more positive voltages. The effect of K(+) could be mimicked by additions of choline or N-methyl D-glucamine and could be explained by unspecific screening of the negative surface charge. Fitting the dependence of voltage shift on K(+) concentration to the Gouy-Chapman model yields a surface charge density of 0.36 +/- 0.05 e(-)/nm(2). Negative surface potential also tended to increase the local concentration of permeable ions (K(+)), resulting in anomalously high single-channel conductance, approximately 200 pS in 10 mM KCl. An increase of ionic strength due to addition of impermeable cations greatly reduced the unitary conductance. Large positive shift of the SV channel voltage dependence, caused by physiological (0.5 mM) free vacuolar Ca(2+), was partly ameliorated by increasing luminal K(+). We interpreted these results as follows: K(+)induced a reduction of surface potential, hence i) causing a positive shift of the voltage dependence and ii) a dilution of Ca(2+) in the membrane vicinity, thus reducing the inhibitory effect of vacuolar Ca(2+) and causing a negative shift of the SV channel voltage dependence, with a sum of the two shifts being negative.

Regulation of Kv4.3 currents by Ca2+/calmodulin-dependent protein kinase II.

The voltage-dependent K+ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K+ currents, including the transient outward current in the heart (Ito) and A currents (IA) in neuronal and smooth muscle preparations. Recent studies have shown that Ito in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate Kv4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged Kv4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel.

Characterization of an ultrarapid delayed rectifier potassium channel involved in canine atrial repolarization.

1. Depolarizing pulses positive to 0 mV elicit a transient outward current (Ito) and a sustained 'pedestal' current in canine atrial myocytes. The pedestal current was highly sensitive to 4-aminopyridine (4-AP) and TEA, with 50% inhibitory concentrations (EC50) of 5.3 +/- 0.7 and 307 +/- 25 microM, respectively. When the pedestal current was separated from Ito with prepulses or by studying current sensitive to 10 mM TEA, it showed very rapid activation and deactivation. We therefore designated the current IKur,d, for 'ultrarapid delayed rectifier, dog'. IKur,d inactivation was bi-exponential, with mean time constants of 609 +/- 91 and 5563 +/- 676 ms during a 20 s pulse to +40 mV. 2. The reversal potential of IKur,d tail currents are dependent on extracellular potassium concentration ([K+]o; slope, 54.7 mV decade-1). The envelope of tails test was satisfied and the current inwardly rectified at > or = +40 mV. The current was insensitive to E-4031, dendrotoxin and chloride substitution, but was inhibited by barium, with an EC50 of 1.65 mM. Lanthanum ions caused a positive shift in voltage dependence without producing direct inhibition. 3. Single-channel activity was observed in cell-attached, inside-out and outside-out patches. Upon depolarization from -50 to +30 mV, single channels had similar time constants and [K+]o dependence to whole-cell current. Channel open probability (Po) increased with depolarization in a saturable fashion and the Po-voltage relation had a half-activation voltage and slope factor similar to whole-cell IKur,d. 4. Unitary channel current was linearly related to depolarization potential to +40 mV; at more positive potentials, inward rectification occurred. The unitary conductance was 20.3 and 35.5 pS for an [K+]o of 5.4 and 130 mM, respectively. Single-channel activity was strongly inhibited by 50 microM 4-AP or 10 mM TEA. Both 4-AP and TEA decreased open time, suggesting open-channel block. 5. Selective inhibition of IKur,d with 50 microM 4-AP or 0.3-5 mM TEA prolonged canine atrial action potentials, indicating that IKur,d contributes to canine atrial repolarization. The single-channel and macroscopic properties of IKur,d have many similarities to those of currents carried by Kv3.1 cloned channels and our findings thus suggest a possible role for Kv3.1 channels in cardiac repolarization.

Biochemical separation of delayed rectifier currents in frog short skeletal muscle fibres.

Frog short (1-1.5 mm) skeletal muscle fibres in sucrose hypertonic Ringer at 5 degrees C were voltage clamped employing a two-electrode technique. Decreasing pH from 7.0 to 5.0 dramatically increased the rate of turn on of the slow delayed rectifier (GK,s) current, so that it became similar to the rapidly activated form. The accelerating effects of decreased pH upon slow current kinetics had a pKa of 5.8. Decreased pH also appeared to shift the voltage dependence of fast and GK,s gating to more positive potentials. In addition, a decrease in pH from 7.0 to 5.0 shifted the reversal potential of GK,s by over 11 mV in the positive direction. GK,s was selectively and irreversibly abolished by applying 1 mM-diethylpyrocarbonate (DEP), a histidine reagent. This effect occurred even after GK,s had been accelerated by low pH. DEP had no effect upon fast delayed rectifier current except for a small positive shift in voltage dependence. Application of 1 or 2 mM-N-ethylmaleimide, a sulphydryl reagent, depressed the fast delayed rectifier while sparing the GK,s currents. However, the muscle fibres also developed markedly increased leakage currents.