Half-maximal Activation Voltage Research Articles

Functional Characterization of the A414G Loss-of-Function Mutation in HCN4 Associated with Sinus Bradycardia

Patients carrying the heterozygous A414G mutation in the HCN4 gene, which encodes the HCN4 protein, demonstrate moderate to severe bradycardia of the heart. Tetramers of HCN4 subunits compose the ion channels in the sinus node that carry the hyperpolarization-activated ‘funny’ current (If), also named the ‘pacemaker current’. If plays an essential modulating role in sinus node pacemaker activity. To assess the mechanism by which the A414G mutation results in sinus bradycardia, we first performed voltage clamp measurements on wild-type (WT) and heterozygous mutant HCN4 channels expressed in Chinese hamster ovary (CHO) cells. These experiments were performed at physiological temperature using the amphotericin-perforated patch-clamp technique. Next, we applied the experimentally observed mutation-induced changes in the HCN4 current of the CHO cells to If of the single human sinus node cell model developed by Fabbri and coworkers. The half-maximal activation voltage V1/2 of the heterozygous mutant HCN4 current was 19.9 mV more negative than that of the WT HCN4 current (p < 0.001). In addition, the voltage dependence of the heterozygous mutant HCN4 current (de)activation time constant showed a −11.9 mV shift (p < 0.001) compared to the WT HCN4 current. The fully-activated current density, the slope factor of the activation curve, and the reversal potential were not significantly affected by the heterozygous A414G mutation. In the human sinus node computer model, the cycle length was substantially increased, almost entirely due to the shift in the voltage dependence of steady-state activation, and this increase was more prominent under vagal tone. The introduction of a passive atrial load into the model sinus node cell further reduced the beating rate, demonstrating that the bradycardia of the sinus node was even more pronounced by interactions between the sinus node and atria. In conclusion, the experimentally identified A414G-induced changes in If can explain the clinically observed sinus bradycardia in patients carrying the A414G HCN4 gene mutation.

Xenon's Sedative Effect Is Mediated by Interaction with the Cyclic Nucleotide-Binding Domain (CNBD) of HCN2 Channels Expressed by Thalamocortical Neurons of the Ventrobasal Nucleus in Mice.

Previous studies have shown that xenon reduces hyperpolarization-activated cyclic nucleotide-gated channels type-2 (HCN2) channel-mediated current (Ih) amplitude and shifts the half-maximal activation voltage (V1/2) in thalamocortical circuits of acute brain slices to more hyperpolarized potentials. HCN2 channels are dually gated by the membrane voltage and via cyclic nucleotides binding to the cyclic nucleotide-binding domain (CNBD) on the channel. In this study, we hypothesize that xenon interferes with the HCN2 CNBD to mediate its effect. Using the transgenic mice model HCN2EA, in which the binding of cAMP to HCN2 was abolished by two amino acid mutations (R591E, T592A), we performed ex-vivo patch-clamp recordings and in-vivo open-field test to prove this hypothesis. Our data showed that xenon (1.9 mM) application to brain slices shifts the V1/2 of Ih to more hyperpolarized potentials in wild-type thalamocortical neurons (TC) (V1/2: -97.09 [-99.56--95.04] mV compared to control -85.67 [-94.47--82.10] mV; p = 0.0005). These effects were abolished in HCN2EA neurons (TC), whereby the V1/2 reached only -92.56 [-93.16- -89.68] mV with xenon compared to -90.03 [-98.99--84.59] mV in the control (p = 0.84). After application of a xenon mixture (70% xenon, 30% O2), wild-type mice activity in the open-field test decreased to 5 [2-10] while in HCN2EA mice it remained at 30 [15-42]%, (p = 0.0006). In conclusion, we show that xenon impairs HCN2 channel function by interfering with the HCN2 CNBD site and provide in-vivo evidence that this mechanism contributes to xenon-mediated hypnotic properties.

Human Sinoatrial Node Pacemaker Activity: Role of the Slow Component of the Delayed Rectifier K+ Current, IKs.

The pacemaker activity of the sinoatrial node (SAN) has been studied extensively in animal species but is virtually unexplored in humans. Here we assess the role of the slowly activating component of the delayed rectifier K+ current (IKs) in human SAN pacemaker activity and its dependence on heart rate and β-adrenergic stimulation. HEK-293 cells were transiently transfected with wild-type KCNQ1 and KCNE1 cDNA, encoding the α- and β-subunits of the IKs channel, respectively. KCNQ1/KCNE1 currents were recorded both during a traditional voltage clamp and during an action potential (AP) clamp with human SAN-like APs. Forskolin (10 µmol/L) was used to increase the intracellular cAMP level, thus mimicking β-adrenergic stimulation. The experimentally observed effects were evaluated in the Fabbri-Severi computer model of an isolated human SAN cell. Transfected HEK-293 cells displayed large IKs-like outward currents in response to depolarizing voltage clamp steps. Forskolin significantly increased the current density and significantly shifted the half-maximal activation voltage towards more negative potentials. Furthermore, forskolin significantly accelerated activation without affecting the rate of deactivation. During an AP clamp, the KCNQ1/KCNE1 current was substantial during the AP phase, but relatively small during diastolic depolarization. In the presence of forskolin, the KCNQ1/KCNE1 current during both the AP phase and diastolic depolarization increased, resulting in a clearly active KCNQ1/KCNE1 current during diastolic depolarization, particularly at shorter cycle lengths. Computer simulations demonstrated that IKs reduces the intrinsic beating rate through its slowing effect on diastolic depolarization at all levels of autonomic tone and that gain-of-function mutations in KCNQ1 may exert a marked bradycardic effect during vagal tone. In conclusion, IKs is active during human SAN pacemaker activity and has a strong dependence on heart rate and cAMP level, with a prominent role at all levels of autonomic tone.

Palmitoylation regulates the magnitude of HCN4-mediated currents in mammalian cells.

The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na+ and Ca2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (IHCN4) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of IHCN4 or the activation slope factor. IHCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics.

Altered voltage-dependence revealed in an inactivation-deficient mutant of Nav1.4 via optogenetic PI(4,5)P2 dephosphorylation.

Voltage-gated sodium (NaV) channels are a key membrane protein of most excitable cells that activate upon depolarization, which initiates an action potential by rapid influx of Na+ ions. Voltage-dependent activation is followed by fast inactivation, which confers the refractory period for excitable tissues. Because the gating processes of NaV channels are mechanically coupled, changes in the fast inactivation process also alter their voltage-dependent activation properties. Most ion channels are regulated by membrane phospholipid phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2), however the PI(4,5)P2 modulation of NaV channels has not been determined. The mutant channel NaV1.4-L435W-L437C-A438W (WCW) presents significantly attenuated fast-inactivation and expresses robustly in Xenopus oocytes. To study the effects of PI(4,5)P2 dephosphorylation on NaV1.4 activation in channels with attenuated fast inactivation, we expressed NaV1.4-WCW in Xenopus oocytes and recorded currents using two-electrode voltage-clamp (TEVC). To dephosphorylate PI(4,5)P2, we co-expressed 4’ or 5’ phospholipid phosphatases that are recruited to the cell membrane upon exposure to blue light (λ=470 nm). 4’ dephosphorylation of PI(4,5)P2 produced an 8 ± 2% increase in peak NaV1.4 WCW channel currents at a depolarizing pulse to −20 mV. Significantly greater channel currents at −15 mV and −25 mV were also observed. Similarly, 5’ dephosphorylation of PI(4,5)P2 resulted in a 15 ± 2% increase in Nav1.4 WCW peak channel currents. Significantly greater channel currents were observed in the voltage range from −20 mV to +20 mV. Although the peak currents were augmented, the half-maximal activation voltage (V1/2) and slope factor (Kact) of the WCW mutant were both unaffected by either dephosphorylation, suggesting that the altered voltage-dependence of this channel mutant is at least partially due to the effect of PI(4,5)P2 dephosphorylation on fast inactivation.

Inhibition of Cav1.4 channels by protons depends on the identity of the α2δ subunit.

In retinal photoreceptors, CaV1.4 channels coassemble with the β2 and α2δ-4 subunits and regulate the release of neurotransmitter. Like other CaV channels, CaV1.4 undergoes a prominent inhibition by extracellular protons with low pH decreasing the maximal current amplitude (Imax) and depolarizing the half maximal activation voltage (Vh). However, the mechanisms underlying this form of regulation remain controversial. Here, we analyzed the role of subunit identity on proton-mediated inhibition of CaV1.4 (coexpressed with β2x13) in transfected HEK 293T cells. In cells cotransfected with α2δ-4, changing the extracellular pH from 8.0 to 6.25 caused a significantly greater inhibition of Imax (ΔIMax) and shift in Vh (ΔVh) (ΔIMax = 89.8 ± 1.1 %, ΔVh = 22.7 ± 1.7 mV) than in cells cotransfected with α2δ-1 (ΔIMax = 64.6 ± 4.9 %, ΔVh = 9.0 ± 1.4 mV) or expressing CaV1.4 alone (ΔIMax = 48.3 ± 3.0 %, ΔVh = 9.4 ± 2.1 mV). Notably, this effect of α2δ-4 was selectively diminished for a C-terminal variant of CaV1.4 channels with hyperpolarized voltage-dependence of activation due to a disease-causing mutation (K1591X) but not alternative splicing (CaV1.4 Δ47). The increase in ΔIMax due to α2δ-4 was significantly lower in cells expressing K1591X (11.0 ± 9.9 %) as compared to CaV1.4 (46.2 ± 0.7 %) and CaV1.4 Δ47 (28.7 ± 2.4 %). We conclude that α2δ-4 potentiates proton-mediated inhibition of CaV1.4 in a manner that is modified by pathological variations in the C-terminal domain of CaV1.4.

Functional Characterization of Knockdown Resistance Mutation L1014S in the German Cockroach, Blattella germanica (Linnaeus)

The effectiveness of pyrethroid insecticides is seriously threatened by knockdown resistance (kdr), which is induced in insects by inherited single-nucleotide polymorphisms in the voltage-gated sodium channel (VGSC) gene. VGSC's L1014F substitution results in the classic kdr mutation, which is found in many pest species. Other substitutions of the L1014 locus, such as L1014S, L1014C, L1014W, and L1014H, were also reported. In 2022, a new amino acid substitute L1014S of Blattella germanica was first discovered in China. We modified the BgNav1-1 sodium channel from cockroaches with the L1014S mutation to study how pyrethroid sensitivity and channel gating were affected in Xenopus oocytes. The L1014S mutation reduced the half-maximal activation voltage (V1/2,act) from -19.0 (wild type) to -15.5 mV while maintaining the voltage dependency of activation. Moreover, the voltage dependence of inactivation in the hyperpolarizing shifts from -48.3 (wild type) to -50.9 mV. However, compared with wild type, the mutation L1014S did not cause a significant shift in the half activation voltage (V1/2,act). Notably, the voltage dependency of activation was unaffected greatly by the L1014S mutation. Tail currents are induced by two types of pyrethroids (1 μM): type I (permethrin, bifenthrin) and type II (deltamethrin, λ-cyhalothrin). All four pyrethroids produced tail currents, and significant differences were found in the percentages of channel modifications between variants and wild types. Further computer modeling showed that the L1014S mutation allosterically modifies pyrethroid binding and action on B. germanica VGSC, with some residues playing a critical role in pyrethroid binding. This study elucidated the pyrethroid resistance mechanism of B. germanica and predicted the residues that may confer the risk of pyrethroid resistance, providing a molecular basis for understanding the resistance mechanisms conferred by mutations at the 1014 site in VGSC.

The Functional Role of Hyperpolarization Activated Current (I f) on Cardiac Pacemaking in Human vs. in the Rabbit Sinoatrial Node: A Simulation and Theoretical Study.

The cardiac hyperpolarization-activated “funny” current (If), which contributes to sinoatrial node (SAN) pacemaking, has a more negative half-maximal activation voltage and smaller fully-activated macroscopic conductance in human than in rabbit SAN cells. The consequences of these differences for the relative roles of If in the two species, and for their responses to the specific bradycardic agent ivabradine at clinical doses have not been systematically explored. This study aims to address these issues, through incorporating rabbit and human If formulations developed by Fabbri et al. into the Severi et al. model of rabbit SAN cells. A theory was developed to correlate the effect of If reduction with the total inward depolarising current (Itotal) during diastolic depolarization. Replacing the rabbit If formulation with the human one increased the pacemaking cycle length (CL) from 355 to 1,139 ms. With up to 20% If reduction (a level close to the inhibition of If by ivabradine at clinical concentrations), a modest increase (~5%) in the pacemaking CL was observed with the rabbit If formulation; however, the effect was doubled (~12.4%) for the human If formulation, even though the latter has smaller If density. When the action of acetylcholine (ACh, 0.1 nM) was considered, a 20% If reduction markedly increased the pacemaking CL by 37.5% (~27.3% reduction in the pacing rate), which is similar to the ivabradine effect at clinical concentrations. Theoretical analysis showed that the resultant increase of the pacemaking CL is inversely proportional to the magnitude of Itotal during diastolic depolarization phase: a smaller If in the model resulted in a smaller Itotal amplitude, resulting in a slower pacemaking rate; and the same reduction in If resulted in a more significant change of CL in the cell model with a smaller Itotal. This explained the mechanism by which a low dose of ivabradine slows pacemaking rate more in humans than in the rabbit. Similar results were seen in the Fabbri et al. model of human SAN cells, suggesting our observations are model-independent. Collectively, the results of study explain why low dose ivabradine at clinically relevant concentrations acts as an effective bradycardic agent in modulating human SAN pacemaking.

State-dependent inhibition of BK channels by the opioid agonist loperamide.

Large-conductance Ca2+-activated K+ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K+ efflux through intestinal BK channels.

Contributions of S- and R-citalopram to the citalopram-induced modulation of the function of Nav1.5 voltage-gated sodium channels

Citalopram, a selective serotonin reuptake inhibitor (SSRI), has been reported to have adverse effects such as cardiotoxicity, including prolongation of the QTc interval. Although citalopram is well known to be a racemic compound comprised of S-citalopram (escitalopram) and R-citalopram, it is still unclear which enantiomer is responsible for cardiotoxicity induced by citalopram. It is also unclear which biomolecule is the target that produces the adverse effect of citalopram. In this study, we investigated whether citalopram, escitalopram and R-citalopram had an electrophysiological effect on Nav1.5 voltage-gated sodium channel (VGSC) current and how their electrophysiological properties affected Nav1.5 VGSC. To examine the effects of the electrophysiological properties of them, whole-cell patch clamp recording was performed using HEK293 cells expressing human Nav1.5 VGSCs. Nav1.5 VGSC current decreased by 60.0 ± 6.3% and 55.1 ± 12.5% under treatment with 100 μM citalopram and escitalopram, respectively. However, 100 μM R-citalopram decreased Nav1.5 VGSC current by only 36.2 ± 8.7%. In addition, treatment with 100 μM citalopram and escitalopram changed the voltage-dependence of activation and induced a negative shift of the voltage of half-maximal activation compared to 100 μM R-citalopram. In contrast, treatment with 100 μM citalopram and escitalopram, but not R-citalopram, changed the voltage-dependence of inactivation, and the voltage at half-maximal inactivation slightly shifted toward negative potential. These results suggest that the adverse cardiac effect produced by citalopram might result from modification of the electrophysiological properties of Nav1.5 VGSCs, and escitalopram might contribute more to this adverse effect than R-citalopram.

Electrophysiological characterization of the modified hERGT potassium channel used to obtain the first cryo-EM hERG structure.

The voltage‐gated hERG (human‐Ether‐à‐go‐go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss‐of‐function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain‐of‐function mutations lead to short QT syndrome. A recent open channel cryo‐EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure‐function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full‐length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole‐cell measurements of hERG current (IhERG) were made at 37°C from HEK 293 cells expressing wild‐type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end‐pulse nor tail IhERG density significantly differed between WT and hERGT. However, the IhERG deactivation rate was significantly slower for hERGT. Half‐maximal activation voltage (V0.5) was positively shifted for hERGT by ~+8 mV (p < .05 versus WT), without significant change to the activation relation slope factor. Neither the voltage dependence of inactivation, nor time course of development of inactivation significantly differed between WT and hERGT, but recovery of IhERG from inactivation was accelerated for hERGT (p < .05 versus WT). Steady‐state “window” current was positively shifted for hERGT with a modest increase in the window current peak. Under action potential (AP) voltage clamp, hERGT IhERG showed modestly increased current throughout the AP plateau phase with a significant increase in current integral during the AP. The observed consequences for hERGT IhERG of deletion of the two cytoplasmic regions may reflect changes to electrostatic interactions influencing the voltage sensor domain.

Molecular mechanism of BK channel activation by the smooth muscle relaxant NS11021.

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at -200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than -60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

Impact of voltage-gated Na + channel biophysical properties on action potential upstroke

The main determinant of conduction velocity is the AP maximum depolarization rate (dEm/dtmax), which depends on the voltage-gated Na+ channels biophysical properties. Here we investigated age-dependent differences in whole-cell Na+ current (INa) and dEm/dtmax in myocytes isolated from neonatal and adult Wistar rats. The impact of Na+ channel biophysical properties on AP overshoot was evaluated with an AP model developed with measured whole-cell INa, Ca2+, and transient outward and delayed rectifier K+ currents from immature cells, described according to Hodgkin-Huxley kinetics. Even though INa density was 2-fold greater in neonatal myocytes (−71.9 ± 35.5, n = 11; 33.3 ± 1 6.7 pA/pF in adults, n = 6; p < .01), dEm/dtmax was not, and tended to be lower in neonates (84.2 ± 35.8 V/s, n = 12) than in adults (100.3 ± 44.4 V/s, n = 7; p = .40). The half-maximal activation voltage (E1/2) was less negative (−31.5 ± 1.7 mV vs. -44.4 ± 6.0 mV; p < .001), and the activation curve had greater slope (k: 7.8 ± 1.2 mV vs. 4.4 ± 0.6 mV; p < .001) in immature than in adult cells. A positive shift was also observed in the steady-state inactivation curve in neonates (E1/2: −76.2 ± 7.8 mV vs. -85.6 ± 4.1 mV; p < .05). The dEm/dtmax of the simulated AP in neonatal myocyte was 81 V/s. However, when the activation and inactivation equations were adjusted to reproduce E1/2 and k values estimated in adult cells, dEm/dtmax increased to 96 V/s, close to the experimental value in adults. Therefore, it seems that, in addition to INa density, voltage-dependence of Na+ channel activation and inactivation may exert marked influence on depolarization rate, and AP propagation velocity.

Molecular Mechanism of BK Channel Activation by the Smooth Muscle Relaxant NS11021

Large-conductance Ca2+-activated K+ channels (BK channels) are activated by cytosolic calcium and depolarized membrane potential under physiological conditions. Thus, these channels control electrical excitability in neurons and smooth muscle by gating K+ efflux and hyperpolarizing the membrane in response to Ca2+ signaling. Altered BK channel function has been linked to epilepsy, dyskinesia, and other neurological deficits in humans, making these channels a key target for drug therapies. To gain insight into mechanisms underlying pharmacological modulation of BK channel gating, here we studied mechanisms underlying activation of BK channels by the biarylthiourea derivative, NS11021, which acts as a smooth muscle relaxant. We observe that increasing NS11021 shifts the half-maximal activation voltage for BK channels toward more hyperpolarized voltages, in both the presence and nominal absence of Ca2+, suggesting that NS11021 facilitates BK channel activation primarily by a mechanism that is distinct from Ca2+ activation. 30 µM NS11021 slows the time course of BK channel deactivation at -200 mV by ∼10-fold compared with 0 µM NS11021, while having little effect on the time course of activation. This action is most pronounced at negative voltages, at which the BK channel voltage sensors are at rest. Single-channel kinetic analysis further shows that 30 µM NS11021 increases open probability by 62-fold and increases mean open time from 0.15 to 0.52 ms in the nominal absence of Ca2+ at voltages less than -60 mV, conditions in which BK voltage sensors are largely in the resting state. We could therefore account for the major activating effects of NS11021 by a scheme in which the drug primarily shifts the pore-gate equilibrium toward the open state.

Involvement of p75NTR in the effects of Aβ on L-type Ca2+ channel in cultured neuronal networks

Ca2+ overload in neurons has been implicated in Alzheimer's Disease (AD). Upregulation of Ca2+ through L-type Ca2+ channels was known to be involved in the neurodegeneration induced by amyloid-β (Aβ) peptides in AD. However, little is known about the mechanism by which upregulation of L-type Ca2+ channel currents is linked to Aβ-induced neuronal toxicity. In the present study, we found that the L-type Ca2+ current in transgenic AD mice (Tg2576) neurons is greater than in wild-type (WT) neurons, and this Ca2+ channel current change were rescued in Tg2576/p75NTR+/− (p75 neurotrophin receptor) neurons. We further examined the changes in the gating of L-type Ca2+ channels following Aβ42 treatment, and the results showed that the L-type Ca2+ channel current was significantly increased by Aβ42 treatment in WT hippocampal neurons. Blocking or decreasing the expression of p75NTR eliminated the influence of Aβ42 on the L-type Ca2+ channel current in WT hippocampal neurons. We also evaluated how Aβ42 affected the voltage-dependent activation and inactivation of L-type Ca2+ channels in cultured WT neurons. The results indicated that the half-maximal activation voltage (V1/2) was left shifted, and the half-inactivation voltage (V1/2) displayed a right shift in neuron treated by Aβ42. Decreasing the expression of p75NTR eliminated the effect of Aβ42 on voltage-dependent activation and inactivation of the L-type Ca2+ channel. These results indicate that Aβ42 changes L-type Ca2+ channel currents by modulating the channel's activation and inactivation dynamics, while decreasing p75NTR expression can remove this effect.

3'-O-Methylorobol Inhibits the Voltage-Gated Sodium Channel Nav1.7 with Anti-Itch Efficacy in A Histamine-Dependent Itch Mouse Model.

An itch is a clinical complication that affects millions of patients. However, few treatment options are available. The voltage-gated sodium channel Nav1.7 is predominantly expressed in peripheral sensory neurons and is responsible for the rising phase of action potentials, thereby mediating nociceptive conduction. A gain-of-function mutation of Nav1.7 results in the hyperexcitability of sensory neurons and causes the inherited paroxysmal itch. Conversely, a monoclonal antibody that selectively inhibits Nav1.7 is able to effectively suppress the histamine-dependent itch in mice. Therefore, Nav1.7 inhibitors may possess the potential to relieve the itch. In the present study, using whole-cell voltage-clamp recordings, we demonstrated that 3’-O-methylorobol inhibited Na+ currents in Nav1.7-CHO cells and tetrodotoxin-sensitive Na+ currents in mouse dorsal root ganglion (DRG) neurons with IC50 (half-maximal inhibitory concentration) values of 3.46 and 6.60 μM, respectively. 3’-O-methylorobol also suppressed the tetrodotoxin-resistant Na+ currents in DRG neurons, though with reduced potency (~43% inhibition at 30 µM). 3’-O-methylorobol (10 µM) affected the Nav1.7 by shifting the half-maximal voltage (V1/2) of activation to a depolarizing direction by ~6.76 mV, and it shifted the V1/2 of inactivation to a hyperpolarizing direction by ~16.79 mV. An analysis of 3’-O-methylorobol activity toward an array of itch targets revealed that 3’-O-methylorobol was without effect on histamine H1 receptor, TRPV1, TRPV3, TRPV4, TRPC4 and TRPM8. The intrathecal administration of 3’-O-methylorobol significantly attenuated compound 48/80-induced histamine-dependent spontaneous scratching bouts and the expression level of c-fos in the nuclei of spinal dorsal horn neurons with a comparable efficacy to that of cyproheptadine. Our data illustrated the therapeutic potential for 3’-O-methylorobol for histamine-dependent itching, and the small molecule inhibition of Nav1.7 may represent a useful strategy to develop novel therapeutics for itching.

The C-terminal HCN4 variant P883R alters channel properties and acts as genetic modifier of atrial fibrillation and structural heart disease

Atrial fibrillation (AF) is the most frequent sustained arrhythmia and can lead to structural cardiac changes, known as tachycardia-induced cardiomyopathy (TIC). HCN4 is implicated in spontaneous excitation of the sinoatrial node, while channel dysfunction has been associated with sinus bradycardia, AF and structural heart disease. We here asked whether HCN4 mutations may contribute to the development of TIC, as well.Mutation scanning of HCN4 in 60 independent patients with AF and suspected TIC followed by panel sequencing in carriers of HCN4 variants identified the HCN4 variant P883R [minor allele frequency (MAF): 0,88%], together with the KCNE1 variant S38G (MAF: 65%) in three unrelated patients. Family histories revealed additional cases of AF, sudden cardiac death and cardiomyopathy. Patch-clamp recordings of HCN4-P883R channels expressed in HEK293 cells showed remarkable alterations of channel properties shifting the half-maximal activation voltage to more depolarized potentials, while channel deactivation was faster compared to wild-type (WT). Co-transfection of WT and mutant subunits, resembling the heterozygous cellular situation of our patients, revealed significantly higher current densities compared to WT.In conclusion HCN4-P883R may increase ectopic trigger and maintenance of AF by shifting the activation voltage of If to more positive potentials and producing higher current density. Together with the common KCNE1 variant S38G, previously proposed as a genetic modifier of AF, HCN4-P883R may provide a substrate for the development of AF and TIC.

S-Acylation controls functional coupling of BK channel pore-forming α-subunits and β1-subunits

The properties and physiological function of pore-forming α-subunits of large conductance calcium- and voltage-activated potassium (BK) channels are potently modified by their functional coupling with regulatory subunits in many tissues. However, mechanisms that might control functional coupling are very poorly understood. Here we show that S-acylation, a dynamic post-translational lipid modification of proteins, of the intracellular S0–S1 loop of the BK channel pore-forming α-subunit controls functional coupling to regulatory β1-subunits. In HEK293 cells, α-subunits that cannot be S-acylated show attenuated cell surface expression, but expression was restored by co-expression with the β1-subunit. However, we also found that nonacylation of the S0–S1 loop reduces functional coupling between α- and β1-subunits by attenuating the β1-subunit-induced left shift in the voltage for half-maximal activation. In mouse vascular smooth muscle cells expressing both α- and β1-subunits, BK channel α-subunits were endogenously S-acylated. We further noted that S-acylation is significantly reduced in mice with a genetic deletion of the palmitoyl acyltransferase (Zdhhc23) that controls S-acylation of the S0–S1 loop. Genetic deletion of Zdhhc23 or broad-spectrum pharmacological inhibition of S-acylation attenuated endogenous BK channel currents independently of changes in cell surface expression of the α-subunit. We conclude that functional effects of S-acylation on BK channels depend on the presence of β1-subunits. In the absence of β1-subunits, S-acylation promotes cell surface expression, whereas in its presence, S-acylation controls functional coupling. S-Acylation thus provides a mechanism that dynamically regulates the functional coupling with β1-subunits, enabling an additional level of conditional, cell-specific control of ion-channel physiology.

BmK AEP, an Anti-Epileptic Peptide Distinctly Affects the Gating of Brain Subtypes of Voltage-Gated Sodium Channels.

BmK AEP, a scorpion peptide purified form the venom of Buthus martensii Karsch, has been reported to display anti-epileptic activity. Voltage-gated sodium channels (VGSCs) are responsible for the rising phase of action potentials (APs) in neurons and, therefore, controlling neuronal excitability. To elucidate the potential molecular mechanisms responsible for its anti-epileptic activity, we examined the influence of BmK AEP on AP firing in cortical neurons and how BmK AEP influences brain subtypes of VGSCs (Nav1.1–1.3 and Nav1.6). BmK AEP concentration-dependently suppresses neuronal excitability (AP firing) in primary cultured cortical neurons. Consistent with its inhibitory effect on AP generation, BmK AEP inhibits Na+ peak current in cortical neurons with an IC50 value of 2.12 µM by shifting the half-maximal voltage of activation of VGSC to hyperpolarized direction by ~7.83 mV without affecting the steady-state inactivation. Similar to its action on Na+ currents in cortical neurons, BmK AEP concentration-dependently suppresses the Na+ currents of Nav1.1, Nav1.3, and Nav1.6, which were heterologously expressed in HEK-293 cells, with IC50 values of 3.20, 1.46, and 0.39 µM with maximum inhibition of 82%, 56%, and 93%, respectively. BmK AEP shifts the voltage-dependent activation in the hyperpolarized direction by ~15.60 mV, ~9.97 mV, and ~6.73 mV in Nav1.1, Nav1.3, and Nav1.6, respectively, with minimal effect on steady-state inactivation. In contrast, BmK AEP minimally suppresses Nav1.2 currents (~15%) but delays the inactivation of the channel with an IC50 value of 1.69 µM. Considered together, these data demonstrate that BmK AEP is a relatively selective Nav1.6 gating modifier which distinctly affects the gating of brain subtypes of VGSCs.

The Role of HCN Domain in the Function of HCN Channels

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels generate funny currents (If) in the heart and hyperpolarization-activated currents (Ih) in the brain. HCN channels are assembled from four subunits, with each subunit containing six transmembrane segments (S1-S6) and a cyclic-nucleotide binding (CNB) domain in the C-terminal region linked to the pore-forming transmembrane segment with a C-linker. Recent cryo-EM structure of HCN1 channels (Lee et al., Cell, 2017, 168:111-120) identified a stretch of 45 amino acids directly preceding the S1 transmembrane segment as a 3-alpha-helical structural motif conserved in all four mammalian HCN channel isoforms. The 3-alpha-helical domain, dubbed HCN domain, forms direct interactions with the S4 transmembrane segment in the voltage sensor from the same subunit and the C-linker/CNB domain from the adjacent subunit. Here we explored the role of the HCN domain in the function of HCN channels. Deletion of the HCN domain abolished currents from HCN channels expressed in Xenopus laevis oocytes. Mutant HCN channels with alanines substituted for the interacting residues between the HCN domain and the C-linker/CNB domain gave rise to hyperpolarization-activated currents with the half-maximal activation voltage (V1/2) similar to the V1/2 of the wild-type HCN channels. Taken together our observations point towards the role of the HCN domain in the functional assembly of HCN channels.