Tail Current Decay Research Articles

A mutational and modeling study of a binding pocket for IKs activators between KCNQ1 and KCNE1.

The IKs current formed by the co-assembly of KCNQ1 and KCNE1 plays an important role in cardiac repolarization. Loss-of-function mutations in KCNQ1 and KCNE1 are linked to LQT syndrome. Mefenamic acid (Mef) an NSAID, and DIDS a stilbene, are known to enhance IKs currents. The present study explores in detail the role of previously identified K41-KCNE1 in Mef action as well as uncovering the KCNQ1/KCNE1 binding site(s) of Mef and DIDS. In silico experiments of drug docking on a model of IKs channel and molecular dynamic (MD) simulations were used to map critical KCNQ1 and KCNE1 residues, followed by site-directed mutagenesis and functional analysis using whole cell patch clamp. Docking procedures and MD simulations show that Mef and DIDS bind between extracellular KCNE1 residues, specifically to the subunits of the external S6 helix; neighbouring S1; and the pore turret of the opposing subunit. Mef transforms the slowly activating WT IKs current (4:4 EQ) into a current with an almost linear waveform, which causes a left shift in activation V1/2 and inhibits tail current decay at −40 mV. These effects are prevented by the K41C-KCNE1 mutation in a stoichiometric manner. MD simulations and mutagenesis scanning identified three critical residues for Mef binding: W323-KCNQ1; Y46-KCNE1; and K41-KCNE1 in which Mef effects are greatly diminished. The effect of DIDS on WT-EQ are less pronounced than Mef, and K41C-KCNE1 was unable to fully prevent effects of DIDS. Our results show that both activators bind in the same general channel region but depend on different critical residues. These findings provide insights into a common drug-induced binding site, suggesting that the chemical structure of either compound could serve as a starting point for the development of IKs agonists.

The I Ks Ion Channel Activator Mefenamic Acid Requires KCNE1 and Modulates Channel Gating in a Subunit-Dependent Manner.

The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (I Ks ), which is partly responsible for cardiomyocyte repolarization and physiologic shortening of the cardiac action potential. Mefenamic acid, a nonsteroidal anti-inflammatory drug, has been identified as an I Ks activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid's effect on I Ks Using whole-cell patch clamp, we show that mefenamic acid enhances I Ks activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating I Ks current into an almost linear current with instantaneous onset and slowed tail current decay, sensitive to the I Ks blocker (3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy) chroman-4-yl]-N-methylmethanesulfonamide (HMR1556). Both single channels, which reveal no change in the maximum conductance, and whole-cell studies, which reveal a dramatically altered conductance-voltage relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the I Ks channel and shifts channel gating kinetics toward more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose and frequency dependence of mefenamic acid action on I Ks channels. Mutational analysis showed that mefenamic acid's effect on I Ks required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all I Ks channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT: The channels which generate the cardiac slow delayed rectifier K+ current (I Ks ) are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by I Ks in heartbeat regulation, enhancing I Ks current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most I Ks activators, unfortunately, only work on KCNQ1 alone and not the physiologically relevant I Ks channel. We have demonstrated that mefenamic acid can enhance I Ks in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.

Mutations of two acidic residues at the cytoplasmic end of segment IIIS6 of an insect sodium channel have distinct effects on pyrethroid resistance

Mutations in sodium channels are known to confer knockdown resistance to pyrethroid insecticides, such as permethrin and cypermethrin, in various agricultural pests and disease vectors. Double mutations, D3i28V and E3i32G, were detected in cypermethrin-resistant Helicoverpa armigera and Heliothis virescens populations. However, the role of the two mutations in pyrethroid resistance remains unclear. In this study, we introduced the mutations into the cockroach sodium channel, BgNav1-1a, and examined their effects on channel gating and pyrethroid sensitivity in Xenopus oocytes. D3i28V alone and the double mutation, D3i28V/E3i32G, shifted the voltage dependence of activation in the depolarizing direction by 15 mV and 20 mV, respectively, whereas E3i32G had no significant effect. D3i28V reduced the amplitude of tail currents induced by permethrin and NRDC 157 (Type I pyrethroids) and deltamethrin and cypermethrin (Type II pyrethroids), whereas E3i32G alone had no effect. Intriguingly, the amplitude of Type II pyrethroid-induced tail current from D3i28V/E3i32G channels was similar to that of BgNav1-1a channels, but the decay of the tail currents was accelerated. Such effects were not observed for Type I pyrethroid-induced tail currents. Computational analysis based on the model of dual pyrethroid receptors on insect sodium channels predicted D3i28V and E3i32G exert their effects on channel gating and pyrethroid action via allosteric mechanisms. Our results not only illustrate the distinct effect of the D3i28V/E3i32G double mutations on Type I vs. Type II pyrethroids, but also reinforce the concept that accelerated decay of tail currents can be an effective mechanism of pyrethroid resistance to Type II pyrethroids.

Serum albumin attenuates the open-channel blocking effects of propofol on the human Kv1.5 channel

The intravenous anesthetic propofol modulates various ion channel functions. It is generally accepted that approximately 98% of propofol binds to blood constituents and that the free (unbound) drug preferentially affects target proteins including ion channels. However, modulatory effects of propofol on ion channels have not been previously explored in the presence of serum albumin. This study was designed to investigate the effects of serum albumin on the blocking action of propofol on the human Kv1.5 (hKv1.5) current. Whole-cell patch-clamp method was used to record the hKv1.5 channel current, heterologously expressed in Chinese hamster ovary cells, in the absence and presence of bovine serum albumin (BSA). Propofol induced a time-dependent decline of the hKv1.5 current during depolarizing steps and slowed the time course of tail current decay upon repolarization, supporting that propofol acts as an open-channel blocker. This blocking effect was reversible and concentration-dependent with an IC50 of 62.9±3.1μM (n = 6). Bath application of 1% BSA markedly reduced the blocking potency of propofol on hKv1.5 current (IC50 of 1116.0±491.4μM; n = 6). However, in the presence of BSA, the propofol-induced inhibition of hKv1.5 current was also accompanied by a gradual decline of activated current during depolarization and deceleration of deactivating tail current upon repolarization. The presence of BSA greatly attenuated the blocking potency of propofol on hKv1.5 channel without affecting the mode of action of propofol on the channel. Serum albumin thus appears to bind to propofol and thereby reducing effective concentrations of the drug for inhibition of hKv1.5 channel.

Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus.

CaV1.1 acts as both the voltage sensor that triggers excitation-contraction coupling in skeletal muscle and as an L-type Ca(2+) channel. It has been proposed that, after its posttranslational cleavage, the distal C terminus of CaV1.1 remains noncovalently associated with proximal CaV1.1, and that tethering of protein kinase A to the distal C terminus is required for depolarization-induced potentiation of L-type Ca(2+) current in skeletal muscle. Here, we report that association of the distal C terminus with proximal CaV1.1 cannot be detected by either immunoprecipitation of mouse skeletal muscle or by colocalized fluorescence after expression in adult skeletal muscle fibers of a CaV1.1 construct labeled with yellow fluorescent protein (YFP) and cyan fluorescent protein on the N and C termini, respectively. We found that L-type Ca(2+) channel activity was similar after expression of constructs that either did (YFP-CaV1.11860) or did not (YFP-CaV1.11666) contain coding sequence for the distal C-terminal domain in dysgenic myotubes null for endogenous CaV1.1. Furthermore, in response to strong (up to 90 mV) or long-lasting prepulses (up to 200 ms), tail current amplitudes and decay times were equally increased in dysgenic myotubes expressing either YFP-CaV1.11860 or YFP-CaV1.11666, suggesting that the distal C-terminal domain was not required for depolarization-induced potentiation. Thus, our experiments do not support the existence of either biochemical or functional interactions between proximal CaV1.1 and the distal C terminus.

Divergent actions of the pyrethroid insecticides S-bioallethrin, tefluthrin, and deltamethrin on rat Na v1.6 sodium channels

We expressed rat Na v1.6 sodium channels in combination with the rat β 1 and β 2 auxiliary subunits in Xenopus laevis oocytes and evaluated the effects of the pyrethroid insecticides S-bioallethrin, deltamethrin, and tefluthrin on expressed sodium currents using the two-electrode voltage clamp technique. S-Bioallethrin, a type I structure, produced transient modification evident in the induction of rapidly decaying sodium tail currents, weak resting modification (5.7% modification at 100 μM), and no further enhancement of modification upon repetitive activation by high-frequency trains of depolarizing pulses. By contrast deltamethrin, a type II structure, produced sodium tail currents that were ~ 9-fold more persistent than those caused by S-bioallethrin, barely detectable resting modification (2.5% modification at 100 μM), and 3.7-fold enhancement of modification upon repetitive activation. Tefluthrin, a type I structure with high mammalian toxicity, exhibited properties intermediate between S-bioallethrin and deltamethrin: intermediate tail current decay kinetics, much greater resting modification (14.1% at 100 μM), and 2.8-fold enhancement of resting modification upon repetitive activation. Comparison of concentration–effect data showed that repetitive depolarization increased the potency of tefluthrin ~ 15-fold and that tefluthrin was ~ 10-fold more potent than deltamethrin as a use-dependent modifier of Na v1.6 sodium channels. Concentration–effect data from parallel experiments with the rat Na v1.2 sodium channel coexpressed with the rat β 1 and β 2 subunits in oocytes showed that the Na v1.6 isoform was at least 15-fold more sensitive to tefluthrin and deltamethrin than the Na v1.2 isoform. These results implicate sodium channels containing the Na v1.6 isoform as potential targets for the central neurotoxic effects of pyrethroids.

Jingzhaotoxin-IX, a novel gating modifier of both sodium and potassium channels from Chinese tarantula Chilobrachys jingzhao

Tarantula Chilobrachys jingzhao is one of the most venomous species distributed in China. In this study, we have isolated and characterized a novel neurotoxin named Jingzhaotoxin-IX (JZTX-IX) from the venom of the tarantula. JZTX-IX is a C-terminally amidated peptide composed of 35 amino acid residues. The toxin shows 74% sequence identity with CcoTx3 from southeastern Africa tarantula Ceratogyrus cornuatus. JZTX-IX was found to interact with multiple types of ion channels including voltage-gated sodium channels (both tetrodotoxin-resistant and tetrodotoxin-sensitive isoforms) and Kv2.1 channel. The toxin had no effect on delayed rectifier potassium channel Kv1.1, 1.2 and 1.3. JZTX-IX shifted the voltage dependence of channel activation to more positive voltages, but binding of toxin to ion channels was not reversible by extreme depolarization. In addition, JZTX-IX could bias the activities of ion channels towards closed state because the time constant for decay (channel deactivation) of tail currents became faster in the presence of toxin. Taken together with the finding that 10 μM JZTX-IX completely blocked ion channels at resting potential without pulsing, we propose that JZTX-IX is a gating modifier showing low selectivity for ion channel types and trapping voltage sensor at closed state.

Mechanism Of IKr Loss In Mutant T421M-hERG Expressing Rat Ventricular Myocytes

The human ether a-go-go-related gene (hERG) encodes rapidly activating delayed rectifier K+ current (IKr) in heart. Missense mutations in hERG lead to loss in IKr in patients with type 2 long QT (LQT2) syndrome. In native adult rat ventricular myocytes (ARVMs), which intrinsically lack IKr, we studied hERG current (IhERG) by expressing WT or T421M mutation-hERG protein using an adenoviral infection system. The T421M mutation was identified in the S1 transmembrane region of hERG in a 32-yr-old woman with LQT2. Isolated ARVMs were infected with WT or T421M-expressing adenovirus and IhERG was recorded from infected myocytes 4 days after infection. Expression of WT or T421M-hERG in ARVMs produced IhERG with peak tail current (prepulse to 50 mV, tail current recorded at -50 mV) of 9.9 ± 1.7 and 1.6 ± 0.1 pA/pF (mean ± SEM) respectively. Western blot analysis shows that T421M-hERG protein traffics similar to WT-hERG indicated by the presence of 135 and 155 kDa protein. The voltage dependence of activation for the T421M-hERG showed a marked positive shift of 38 mV (V1/2, 24.5 ± 2.3 mV) compared to WT (-13.5 ± 3.0 mV, p<0.05). The deactivation time constants were derived by fitting tail current decay as a double exponential at hyperpolarizing potentials and were 5-10 fold faster for T421M than WT-hERG. We conclude that in native ventricular myocytes, T421M channels traffic normally and undergo minimal voltage-dependent activation during cardiac action potential repolarization, which is a novel mechanism for loss of IKr. Our results emphasize the importance of S1 region in modulating gating properties of hERG channels. Our adenoviral-mediated, over- expression ARVM model may be exploited to study the changes in cardiac action potential waveforms in cardiomyocyte's native background.

Role of N-Terminal Domain and Accessory Subunits in Controlling Deactivation-Inactivation Coupling of Kv4.2 Channels

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2Δ2–10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2Δ2–10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.

A comparison of the effects of veratridine on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in isolated rat dorsal root ganglion neurons

The effects of veratridine have been compared on tetrodotoxin-sensitive (TTXS) and tetrodotoxin-resistant (TTXR) voltage-gated sodium channels (VGSC) in rat dorsal root ganglion neurons. Veratridine caused a dose-dependent decrease in the peak amplitude of both TTXR and TTXS VGSC currents. When exposed to 25 microM veratridine, TTXS currents but not TTXR currents developed a clear persistent component. The deactivation of both TTXS and TTXR currents was slowed, as evidenced by the appearance of slowly decaying tail currents in voltage clamp records, but the slowing of deactivation was nearly 100 times greater for TTXS than for TTXR currents. Properties of the veratridine-modified VGSCs, derived from an analysis of the slow tail currents, were similar for both TTXS and TTXR in that the V50 for activation and the reversal potential were shifted to more negative potentials than control currents and by a similar amount for each. The relatively fast decay of veratridine-modified TTXR tail currents reflects a faster dissociation of veratridine from TTXR than from TTXS VGSCs. This difference probably underlies the lack of effect of veratridine on TTXR VGSCs in cells that are not voltage-clamped and undermines its value as a chemical activator of putative NaV1.8 TTXR channels.

K-aggravated myotonia mutations at residue G1306 differentially alter deactivation gating of human skeletal muscle sodium channels.

Fast inactivation and deactivation gating were compared between wild-type human voltage-gated skeletal muscle sodium channel (hNaV1.4) and potassium-aggravated myotonia (PAM) mutations G1306A, G1306E, and G1306V. Cell-attached macropatches were used to compare wild-type and PAM-gating properties in normal extracellular K+ (4 mM), decreased K+ (1 mM), and increased K+ (10 mM). G1306E/A increased the apparent valence of the conductance (g(V)) curve. Compared to hNaV1.4, the steady-state inactivation (h infinity) curve was depolarized for G1306E/A but hyperpolarized by G1306V, and this mutation increased apparent valence. G1306A/E slowed the rate of current rise towards peak activation. G1306V slowed open-state deactivation, inactivated-state deactivation, and recovery from fast inactivation. G1306A/E abbreviated open-state deactivation at negative commands. These mutants slowed open-state deactivation at more positive commands, at voltages for which fast inactivation might influence tail current decay. G1306E abbreviated recovery delay without affecting recovery rate. Low K+ increased peak current in hNaV1.4 and in G1306V. For G1306E, low K+ increased the rate of entry into fast inactivation, hyperpolarized the g(V) and h(infinity) curves, and increased recovery delay. Biophysical underpinnings of PAM caused by mutations of G1306 thus vary with the specific mutation, and hyperkalemic exacerbation of effects of mutations at this residue are not direct.

Structure–activity relationships for the action of 11 pyrethroid insecticides on rat Nav1.8 sodium channels expressed in Xenopus oocytes

Pyrethroid insecticides bind to voltage-sensitive sodium channels and modify their gating kinetics, thereby disrupting nerve function. This paper describes the action of 11 structurally diverse commercial pyrethroid insecticides on the rat Na v 1.8 sodium channel isoform, the principal carrier of the tetrodotoxin-resistant, pyrethroid-sensitive sodium current of sensory neurons, expressed in Xenopus laevis oocytes. All 11 compounds produced characteristic sodium tail currents following a depolarizing pulse that ranged from rapidly-decaying monoexponential currents (allethrin, cismethrin and permethrin) to persistent biexponential currents (cyfluthrin, cyhalothrin, cypermethrin and deltamethrin). Tail currents for the remaining compounds (bifenthrin, fenpropathrin, fenvalerate and tefluthrin) were monoexponential and decayed with kinetics intermediate between these extremes. Reconstruction of currents carried solely by the pyrethroid-modified subpopulation of channels revealed two types of pyrethroid-modified currents. The first type, found with cismethrin, allethrin, permethrin and tefluthrin, activated relatively rapidly and inactivated partially during a 40-ms depolarization. The second type, found with cypermethrin, cyfluthrin, cyhalothrin, deltamethrin, fenpropathrin and fenvalerate, activated more slowly and did not detectably inactivate during a 40-ms depolarization. Only bifenthrin did not produce modified currents that fit clearly into either of these categories. In all cases, the rate of activation of modified channels was strongly correlated with the rate of tail current decay following repolarization. Modification of Na v 1.8 sodium channels by cyfluthrin, cyhalothrin, cypermethrin and deltamethrin was enhanced 2.3- to 3.4-fold by repetitive stimulation; this effect appeared to result from the accumulation of persistently open channels rather than preferential binding to open channel states. Fenpropathrin was the most effective compound against Na v 1.8 sodium channels from the perspective of either resting or use-dependent modification. When use dependence is taken into account, cypermethrin, deltamethrin and tefluthrin approached the effectiveness of fenpropathrin. The selective expression of Na v 1.8 sodium channels in nociceptive neurons suggests that these channels may be important targets for pyrethroids in the production of paresthesia following dermal exposure.

Regulatory mechanisms and physiological relevance of a voltage-gated H+ channel in murine osteoclasts: phorbol myristate acetate induces cell acidosis and the channel activation.

The voltage-gated H+ channel is a powerful H+ extruding mechanism of osteoclasts, but its functional roles and regulatory mechanisms remain unclear. Electrophysiological recordings revealed that the H+ channel operated on activation of protein kinase C together with cell acidosis. H+ is a key signaling ion in bone resorption. In addition to H+ pumps and exchangers, osteoclasts are equipped with H+ conductive pathways to compensate rapidly for pH imbalance. The H+ channel is distinct in its strong H+ extrusion ability and voltage-dependent gatings. To investigate how and when the H+ channel is available in functional osteoclasts, the effects of phorbol 12-myristate 13-acetate (PMA), an activator for protein kinase C, on the H+ channel were examined in murine osteoclasts generated in the presence of soluble RANKL (sRANKL) and macrophage-colony stimulating factor (M-CSF). Whole cell recordings clearly showed that the H+ current was enhanced by increasing the pH gradient across the plasma membrane (delta(pH)), indicating that the H+ channel changed its activity by sensing delta(pH). The reversal potential (V(rev)) was a valuable tool for the real-time monitoring of delta(pH) in clamped cells. In the permeabilized patch, PMA (10 nM-1.6 microM) increased the current density and the activation rate, slowed decay of tail currents, and shifted the threshold toward more negative voltages. In addition, PMA caused a negative shift of V(rev), suggesting that intracellular acidification occurred. The PMA-induced cell acidosis was confirmed using a fluorescent pH indicator (BCECF), which recovered quickly in a K(+)-rich alkaline solution, probably through the activated H+ channel. Both cell acidosis and activation of the H+ channel by PMA were inhibited by staurosporine. In approximately 80% of cells, the PMA-induced augmentation in the current activity remained after compensating for the delta(pH) changes, implying that both delta(pH)-dependent and -independent mechanisms mediated the channel activation. Activation of the H+ channel shifted the membrane potential toward V(rev). These data suggest that the H+ channel may contribute to regulation of the pH environments and the membrane potential in osteoclasts activated by protein kinase C.

Potentiated L-type Ca2+ channels rectify.

Strong depolarization and dihydropyridine agonists potentiate inward currents through native L-type Ca2+ channels, but the effect on outward currents is less clear due to the small size of these currents. Here, we examined potentiation of wild-type α1C and two constructs bearing mutations in conserved glutamates in the pore regions of repeats II and IV (E2A/E4A-α1C) or repeat III (E3K-α1C). With 10 mM Ca2+ in the bath and 110 mM Cs+ in the pipette, these mutated channels, expressed in dysgenic myotubes, produced both inward and outward currents of substantial amplitude. For both the wild-type and mutated channels, we observed strong inward rectification of potentiation: strong depolarization had little effect on outward tail currents but caused the inward tail currents to be larger and to decay more slowly. Similarly, exposure to DHP agonist increased the amplitude of inward currents and decreased the amplitude of outward currents through both E2A/E4A-α1C and E3K-α1C. As in the absence of drug, strong depolarization in the presence of dihydropyridine agonist had little effect on outward tail currents but increased the amplitude and slowed the decay of inward tail currents. We tested whether cytoplasmic Mg2+ functions as the blocking particle responsible for the rectification of potentiated L-type Ca2+ channels. However, even after complete removal of cytoplasmic Mg2+, (−)BayK 8644 still potentiated inward current and partially blocked outward current via E2A/E4A-α1C. Although zero Mg2+ did not reveal potentiation of outward current by DHP agonist, it did have two striking effects, (a) a strong suppression of decay of both inward and outward currents via E2A/E4A-α1C and (b) a nearly complete elimination of depolarization-induced potentiation of inward tail currents. These results can be explained by postulating that potentiation exposes a binding site in the pore to which an intracellular blocking particle can bind and produce inward rectification of the potentiated channels.

Negative charges in the DIII-DIV linker of human skeletal muscle Na+ channels regulate deactivation gating.

Charge reversing, neutralizing and substituting mutations at D1309 and EE1314,15 in the DIII-DIV linker of the human skeletal muscle sodium channel hNav1.4 were constructed and expressed in Xenopus oocytes. The effects of these mutations on conductance, inactivation and deactivation were determined using on-cell macropatches. D1309R caused a depolarizing shift of the conductance-voltage (g(V)) curve and increased the apparent valency of activation. D1309R and EE1314,15RR increased time to peak activation. D1309R caused a depolarizing shift of the steady-state fast inactivation curve, whereas EE1314,15RR produced a hyperpolarizing shift and decreased the apparent valency. Charge reversal at either D1309 or EE1314,15 slowed open-state fast inactivation and accelerated closed-state fast inactivation. D1309R accelerated recovery from fast inactivation, whereas EE1314,15RR and EE1314,15QQ slowed recovery. Deactivation from the inactivated state was determined by the delay in the onset to recovery from fast inactivation. Recovery delay was abbreviated for D1309R but was prolonged for EE1314,15RR and EE1314,15QQ. Open-state deactivation was determined from the time constant of the decay (tau D) of tail currents. tau D was slowed by D1309R, D1309E, EE1314,15RR and EE1314,15QQ. Our findings suggest an important role in deactivation gating in hNav1.4 for the negative cluster of charge at EE1314,15. These and previous findings suggest that clusters of negatively and positively charged residues in the hNav1.4 DIII-DIV linker differentially regulate the kinetics of fast inactivation.

Interactions between NADPH oxidase-related proton and electron currents in human eosinophils.

1. Proton and electron currents in human eosinophils were studied using the permeabilized-patch voltage-clamp technique, with an applied NH4+ gradient to control pH(i). 2. Voltage-gated proton channels in unstimulated human eosinophils studied with the permeabilized-patch approach had properties similar to those reported in whole-cell studies. 3. Superoxide anion (O2-) release assessed by cytochrome c reduction was compared in human eosinophils and neutrophils stimulated by phorbol myristate acetate (PMA). PMA-stimulated O2 release was more transient and the maximum rate was three times greater in eosinophils. 4. In PMA-activated eosinophils, the H+ current amplitude (I(H)) at +60 mV increased 4.7-fold, activation was 4.0 times faster, deactivation (tail current decay) was 5.4 times slower, the H+ conductance-voltage (g(H)-V) relationship was shifted -43 mV, and diphenylene iodinium (DPI)-inhibitable inward current reflecting electron flow through NADPH oxidase was activated. The data reveal that PMA activates the H+ efflux during the respiratory burst by modulating the properties of H+ channels, not simply as a result of NADPH oxidase activity. 5. The electrophysiological response of eosinophils to PMA resembled that reported in human neutrophils, but PMA activated larger proton and electron currents in eosinophils and the response was more transient. 6. ZnCl2 slowed the activation of H+ currents and shifted the g(H)-V relationship to more positive voltages. These effects occurred at similar ZnCl2 concentrations in eosinophils before and after PMA stimulation. These data are compatible with the existence of a single type of H+ channel in eosinophils that is modulated during the respiratory burst.

Blocking effects of the antiarrhythmic drug propafenone on the HERG potassium channel.

Propafenone has been shown to affect the delayed-rectifier potassium currents in cardiomyocytes of different animal models. In this study we investigated effects and mechanisms of action of propafenone on HERG potassium channels in oocytes of Xenopus laevis with the two-electrode voltage-clamp technique. Propafenone decreased the currents during voltage steps and the tail currents. The block was voltage-dependent and increased with positive going potentials (from 18% block of tail current amplitude at -40 mV to 69% at +40 mV with 100 micromol/l propafenone). The voltage dependence of block could be fitted with the sum of a monoexponential and a linear function. The fractional electrical distance was estimated to be delta=0.20. The block of current during the voltage step increased with time starting from a level of 83% of the control current. Propafenone accelerated the increase of current during the voltage step as well as the decay of tail currents (time constants of monoexponential fits decreased by 65% for the currents during the voltage step and by 37% for the tail currents with 100 micromol/l propafenone). The threshold concentration of propafenone effect was around 1 micromol/l and the concentration of half-maximal block (IC50) ranged between 13 micromol/l and 15 micromol/l for both current components. With high extracellular potassium concentrations, the IC50 value rose to 80 degrees mol/l. Acidification of the extracellular solution to pH 6.0 increased the IC50 value to 123 micromol/l, alkalization to pH 8.0 reduced it to 10 micromol/l and coexpression of the beta-subunit minK had no statistically significant effect on the concentration dependence. In conclusion, propafenone has been found to block HERG potassium channels. The data suggest that propafenone affects the channels in the open state and give some hints for an intracellular site of action.

The V410M mutation associated with pyrethroid resistance in Heliothis virescens reduces the pyrethroid sensitivity of house fly sodium channels expressed in Xenopus oocytes

Some strains of Heliothis virescens carry a novel sodium channel mutation, corresponding to the replacement of Val410 by Met (designated V410M) in the house fly Vssc1 sodium channel, that is genetically and physiologically associated with pyrethroid resistance. To test the functional significance of this mutation, we created a house fly Vssc1 sodium channel containing the V410M mutation by site-directed mutagenesis, expressed wildtype and specifically mutated sodium channels in Xenopus laevis oocytes, and evaluated the effects of the V410M mutation on the functional and pharmacological properties of the expressed channels by two-electrode voltage clamp. The V410M mutation caused depolarizing shifts of ∼9 mV and ∼5 mV in the voltage dependence of activation and steady-state inactivation, respectively, of Vssc1 sodium channels. The V410M mutation also reduced the sensitivity of Vssc1 sodium channels to the pyrethroid cismethrin at least 10-fold and accelerated the decay of cismethrin-induced sodium tail currents. The degree of resistance conferred by the V410M mutation in the present study is sufficient to account for the degree of pyrethroid resistance in H. virescens that is associated with this mutation. Although Val410 is located in a sodium channel segment identified as part of the binding site for batrachotoxin, the V410M mutation did not alter the sensitivity of house fly sodium channels to batrachotoxin. The effects of the V410M mutation on the voltage dependence and cismethrin sensitivity of Vssc1 sodium channels were indistinguishable from those caused by another sodium channel point mutation, replacement of Leu1014 by Phe (L1014F), that is the cause of knockdown resistance to pyrethroids in the house fly. The positions of the V410M and L1014F mutations in models of the tertiary structure of sodium channels suggest that the pyrethroid binding site on the sodium channel α subunit is located at the interface between sodium channel domains I and II.

Kinetic effects of FPL 64176 on L-type Ca2+ channels in cardiac myocytes.

To characterize the effects of the Ca2+ channel agonist FPL 64176 on L-type Ca2+ current in isolated rat ventricular myocytes, certain of its effects were compared with those of a better known agonist, S (-) Bay K 8644. Both drugs enhance currents elicited by depolarizing pulses and enhance and slow the decay of tail currents elicited by subsequent repolarization. Both drugs shift the voltage dependence of activation and of inactivation approximately 10 mV in the negative direction, but FPL 64176 slows the rate of both activation and the decline of Ca2+ current during a depolarization, whereas Bay K 8644 accelerates the rate of current decay under the same conditions. In single channel studies in on-cell recording mode, FPL 64176 produced a great lengthening of the channel open time, produced very long openings when the channels were repolarized after a depolarizing stimulus, and had only modest effects on mean closed times and on first latency distributions. FPL 64176 and Bay K 8644 also had minimal effects on L-type channel "on" gating currents, while the "off" gating currents were slowed, particularly at positive potentials. However, the effects on gating currents were too small to account for the prolonged tails observed in FPL 64176. Once the channel is open, FPL 64176 slows transitions to closed or inactivated channel states.

The delay in recovery from fast inactivation in skeletal muscle sodium channels is deactivation.

1. Using macropatch techniques, we tested the assumption that deactivation underlies the observed delay in the onset to recovery from fast inactivation by comparing open-state deactivation to recovery delay for rat skeletal muscle mutations R1441C and R1441P. 2. Deactivation kinetics from the open state were determined from the exponential decay of tail currents. R1441C and R1441P prolonged open-state deactivation, with the greatest effect produced by R1441P. 3. Delays in the onset to recovery from fast inactivation for R1441P and for R1441C were abbreviated compared to those for rSkM1. Recovery delay was longer in R1441P than R1441C at voltages more negative than -110 mV. Recovery from inactivation exhibited a voltage dependence which, unlike delay, saturated at depolarized voltages. Recovery rate constants were increased to a similar extent for R1441C and R1441P at -150 to -120 mV compared to rSkM1. 4. These results indicate that the delay in the onset to recovery from fast inactivation in skeletal muscle sodium channels is due to deactivation. Lessening of charge immobilization for R1441C and R1441P may contribute to observed biophysical defects underlying the hyperexcitability of muscle fibers containing paramyotonia congenita mutations. The second stage of recovery from fast inactivation may be affected differentially by these mutations.