Human adrenal glomerulosa cells express K2P and GIRK potassium channels that are inhibited by ANG II and ACTH.

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

A- and D-type potassium currents regulate axonal action potential repolarization in midbrain dopamine neurons.

Midbrain dopamine neurons (DANs) regulate various brain functions such as motor control and motivation. Alteration of spiking activities of these neurons could contribute to severe brain disorders including Parkinson's disease and depression. Previous studies showed important roles of somatodendritic voltage-gated K+ channels (Kv) of DANs in governing neuronal excitability and dopamine release. However, it remains largely unclear about the biophysical properties and the function of Kv channels distributed at DAN axons. We performed whole-cell recordings from the axons of DANs in acute mouse midbrain and striatal slices. We detected both rapidly activating/inactivating Kv current (i.e. A-current) and rapidly activating but slowly inactivating current (i.e. D-current) in DAN axons. Pharmacological experiments with channel blockers revealed that these currents are predominantly mediated by Kv1.4 and Kv1.2 subunits, respectively. Blocking these currents could substantially prolong axonal action potentials (APs) via a reduction of their repolarization slope. Together, our results show that Kv channels mediating A- and D-currents shape AP waveforms in midbrain DAN axons, through this regulation they may control dopamine release at the axonal terminals. Therefore, these axonal Kv channels could be drug targets for brain disorders with abnormal dopamine release.

Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes.

Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.

Molecular basis involved in the blocking effect of antidepressant metergoline on C-type inactivation of Kv1.4 channel.

Voltage-gated potassium channels (VGKCs) are transmembrane ion channels specific for potassium. Currently there are nine kinds of VGKCs. Kv1.4 is one of shaker-related potassium channels. It is a representative alpha subunit of potassium channels that can inactivate A type-currents, leading to N pattern inactivation. Inactivation of Kv channels plays an important role in shaping electrical signaling properties of neuronal and muscular cells. The shape of N pattern inactivation can be modified by removing the N-terminal (NT) domain which results in non-inactivated currents and C pattern inactivation. In a previous work, we have reported the regulatory effect of metergoline on Kv1.4 and Nav1.2 channel activity. In the present study, we constructed a mutant of deleted 61 residues from NT of Kv1.4 channels (Kv1.4 Δ2-61) and found that it induced an outward peak and steady-state currents We also studied the modulation effect of metergoline on the activity of this Kv1.4 Δ2-61 mutant channel without having the N-terminal quick inactivation domain. Our results revealed that treatment with metergoline inhibited NT deleted Kv1.4 mutant channel activity in a concentration-dependent manner which was reversible. Interestingly, metergoline treatment induced little effects on the outward peak current in the deleted Kv1.4 mutant channel. However, metergoline treatment conspicuously inhibited steady state currents of Kv1.4 Δ2-61 channels with acceleration current mode. The acceleration of steady-state current of deleted Kv1.4 mutant channel occurred in a concentration-dependent manner. This means that metergoline can accelerate C pattern inactivation of Kv1.4 Δ2-61 channel by acting as an open state dependent channel blocker. We also performed site-directed mutations in V561A and K532Y, also known as C-type inactivation sites. V561A, K532Y, and V561A + K532Y substitution mutants significantly attenuated the acceleration effect of metergoline on C pattern inactivation of hKv1.4 channel currents. In docking modeling study, predicted binding residues for metergoline were analyzed for six amino acids. Among them, the K532 residue known as the C-type inactivation site was analyzed to be a major site of action. Then various mutants were constructed. K532 substitution mutant significantly abolished the effect of metergoline on Kv1.4 currents among various mutants whereas other changes had slight inhibitory effects. Furthermore, we found that metergoline had specificity for Kv1.4, but not for Kv1.5 currents. In addition, the A type current in rat neuronal cell was inhibited and accelerated of inactivation. This result further shows that metergoline might interact with Lys532 residue and then accelerate C pattern inactivation of Kv1.4 channels with channel type specificity. Taken together, these results demonstrate the molecular basis involved in the effect of metergoline, an ergot alkaloid, on human Kv1.4 channel, providing a novel interaction ligand.

Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator.

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 µM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.

Subtype-specific block of voltage-gated K+ channels by µ-conopeptides.

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides µ-SIIIA and µ-PIIIA, thus, are not specific for NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.

Effects of ginseng-spikenard heart-nourishing capsule on inactivation of C-type Kv1.4 potassium channel.

This research is to explore the effects of traditional Chinese medicine Ginseng-spikenard heart-nourishing capsule on the inactivation of c-type Kv1.4 channels (Kv1.4∆N) in Xenopus laevis oocytes with two-electrode voltageclamp technique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4δN channels. During recording, oocytes were continuously perfused with ND96 solution (control group) and solution prepared from Ginseng-spikenard heart-nourishing capsule (experimental group). Results found that, at the command potential of +50 mV, the current of experimental group was reduced to 48.33±4.0% of that in control group. The inactivation time constants in control and experimental groups were 2962.56±175.35 ms and 304.13±36.22ms, respectively (P<0.05, n=7). The recovery time of fKv1.4∆N channel after inactivation in control group and experimental groups was 987±68.39 ms and 1734.15±98.45 ms, respectively (P<0.05, n=5). Ginseng-spikenard heart-nourishing capsule can inhibit the Kv1.4δN channel, which may be one of the mechanisms of underlying antiarrhythmia.

Regulation of Human Kv1.4 Channel Activity by the Antidepressant Metergoline.

Metergoline is an ergot-derived psychoactive drug that is a ligand for various serotonin and dopamine receptors. Little is known about the effect of metergoline on different types of receptors and ion channels. Potassium channels are the most diverse group of ion channels. Kv1.4, a shaker family K channel alpha subunit, is one of a family of voltage gated K channels that mediates transient and rapid inactivating A-type currents and N-type inactivation. We demonstrated previously that metergoline inhibited the activity of neuronal voltage-dependent Na(+) channels in Xenopus laevis oocytes (Acta Pharmacol. Sin., 35, 2014, Lee et al.). In this study, we sought to elucidate the regulatory effects underlying metergoline-induced human Kv1.4 channel inhibition. We used the two electrode voltage-clamp (TEVC) technique to investigate the effect of metergoline on human Kv1.4 channel currents in Xenopus laevis oocytes expressing human Kv1.4 alpha subunits. Interestingly, metergoline treatment also induced inhibition of peak currents in human Kv1.4 channels in a concentration-dependent manner. The IC50 of peak currents of hKv1.4 currents was 3.6±0.6 µM. These results indicate that metergoline might regulate the human Kv1.4 channel activity that is expressed in X. laevis oocytes. Further, this regulation of potassium currents by metergoline might be one of the pharmacological actions of metergoline-mediated psychoactivity.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling.

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

Properties of Slo1 K+ channels with and without the gating ring.

High-conductance Ca(2+)- and voltage-activated K(+) (Slo1 or BK) channels (KCNMA1) play key roles in many physiological processes. The structure of the Slo1 channel has two functional domains, a core consisting of four voltage sensors controlling an ion-conducting pore, and a larger tail that forms an intracellular gating ring thought to confer Ca(2+) and Mg(2+) sensitivity as well as sensitivity to a host of other intracellular factors. Although the modular structure of the Slo1 channel is known, the functional properties of the core and the allosteric interactions between core and tail are poorly understood because it has not been possible to study the core in the absence of the gating ring. To address these questions, we developed constructs that allow functional cores of Slo1 channels to be expressed by replacing the 827-amino acid gating ring with short tails of either 74 or 11 amino acids. Recorded currents from these constructs reveals that the gating ring is not required for either expression or gating of the core. Voltage activation is retained after the gating ring is replaced, but all Ca(2+)- and Mg(2+)-dependent gating is lost. Replacing the gating ring also right-shifts the conductance-voltage relation, decreases mean open-channel and burst duration by about sixfold, and reduces apparent mean single-channel conductance by about 30%. These results show that the gating ring is not required for voltage activation but is required for Ca(2+) and Mg(2+) activation. They also suggest possible actions of the unliganded (passive) gating ring or added short tails on the core.

Ca2+ and K+ channels of normal human adrenal zona fasciculata cells: properties and modulation by ACTH and AngII.

In whole cell patch clamp recordings, we found that normal human adrenal zona fasciculata (AZF) cells express voltage-gated, rapidly inactivating Ca(2+) and K(+) currents and a noninactivating, leak-type K(+) current. Characterization of these currents with respect to voltage-dependent gating and kinetic properties, pharmacology, and modulation by the peptide hormones adrenocorticotropic hormone (ACTH) and AngII, in conjunction with Northern blot analysis, identified these channels as Cav3.2 (encoded by CACNA1H), Kv1.4 (KCNA4), and TREK-1 (KCNK2). In particular, the low voltage-activated, rapidly inactivating and slowly deactivating Ca(2+) current (Cav3.2) was potently blocked by Ni(2+) with an IC50 of 3 µM. The voltage-gated, rapidly inactivating K(+) current (Kv1.4) was robustly expressed in nearly every cell, with a current density of 95.0 ± 7.2 pA/pF (n = 64). The noninactivating, outwardly rectifying K(+) current (TREK-1) grew to a stable maximum over a period of minutes when recording at a holding potential of -80 mV. This noninactivating K(+) current was markedly activated by cinnamyl 1-3,4-dihydroxy-α-cyanocinnamate (CDC) and arachidonic acid (AA) and inhibited almost completely by forskolin, properties which are specific to TREK-1 among the K2P family of K(+) channels. The activation of TREK-1 by AA and inhibition by forskolin were closely linked to membrane hyperpolarization and depolarization, respectively. ACTH and AngII selectively inhibited the noninactivating K(+) current in human AZF cells at concentrations that stimulated cortisol secretion. Accordingly, mibefradil and CDC at concentrations that, respectively, blocked Cav3.2 and activated TREK-1, each inhibited both ACTH- and AngII-stimulated cortisol secretion. These results characterize the major Ca(2+) and K(+) channels expressed by normal human AZF cells and identify TREK-1 as the primary leak-type channel involved in establishing the membrane potential. These findings also suggest a model for cortisol secretion in human AZF cells wherein ACTH and AngII receptor activation is coupled to membrane depolarization and the activation of Cav3.2 channels through inhibition of hTREK-1.

Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes.

AIM: To study the effects of Na(+) channel blocker flecainide and L-type Ca(2+) channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K(+) channel. METHODS: fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K(+) currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion. RESULTS: fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of -41.74 mV and a slow recovery from inactivation (τ=1.90 s at -90 mV). Flecainide and verapamil blocked the currents with IC(50) values of 512.29 ± 56.92 and 260.71 ± 18.50 µmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve. CONCLUSION: The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K(+) channel blockers to treat arrhythmia.

Resonance in neocortical neurons and networks.

Neocortical networks produce oscillations that often correspond to characteristic physiological or pathological patterns. However, the mechanisms underlying the generation of and the transitions between such oscillatory states remain poorly understood. In this study, we examined resonance in mouse layer V neocortical pyramidal neurons. To accomplish this, we employed standard electrophysiology to describe cellular resonance parameters. Bode plot analysis revealed a range of resonance magnitude values in layer V neurons and demonstrated that both magnitude and phase response characteristics of layer V neocortical pyramidal neurons are modulated by changes in the extracellular environment. Specifically, increased resonant frequencies and total inductive areas were observed at higher extracellular potassium concentrations and more hyperpolarised membrane potentials. Experiments using pharmacological agents suggested that current through hyperpolarization-activated cyclic nucleotide-gated channels (I(h) ) acts as the primary driver of resonance in these neurons, with other potassium currents, such as A-type potassium current and delayed-rectifier potassium current (Kv1.4 and Kv1.1, respectively), contributing auxiliary roles. The persistent sodium current was also shown to play a role in amplifying the magnitude of resonance without contributing significantly to the phase response. Although resonance effects in individual neurons are small, their properties embedded in large networks may significantly affect network behavior and may have potential implications for pathological processes.

Effects of diltiazem and propafenone on the inactivation and recovery kinetics of fKv1.4 channel currents expressed in Xenopus oocytes.

AIM: To investigate the effects of diltiazem, an L-type calcium channel blocker, and propafenone, a sodium channel blocker, on the inactivation and recovery kinetics of fKv1.4, a potassium channel that generates the cardiac transient outward potassium current. METHODS: The cRNA for fKv1.4ΔN, an N-terminal deleted mutant of the ferret Kv1.4 potassium channel, was injected into Xenopus oocytes to express the fKv1.4ΔN channel in these cells. Currents were recorded using a two electrode voltage clamp technique. RESULTS: Diltiazem (10 to 1000 µmol/L) inhibited the fKv1.4ΔN channel in a frequency-dependent, voltage-dependent, and concentration-dependent manner, suggesting an open channel block. The IC(50) was 241.04±23.06 µmol/L for the fKv1.4ΔN channel (at +50 mV), and propafenone (10 to 500 µmol/L) showed a similar effect (IC(50)=103.68±10.13 µmol/L). After application of diltiazem and propafenone, fKv1.4ΔN inactivation was bi-exponential, with a faster drug-induced inactivation and a slower C-type inactivation. Diltiazem increased the C-type inactivation rate and slowed recovery in fKv1.4ΔN channels. However, propafenone had no effect on either the slow inactivation time constant or the recovery. CONCLUSION: Diltiazem and propafenone accelerate the inactivation of the Kv1.4ΔN channel by binding to the open state of the channel. Unlike propafenone, diltiazem slows the recovery of the Kv1.4ΔN channel.

[BK(Ca) channel agonist NS1619 and Kv channel antagonist 4-AP on the facial mechanical pain threshold in a rat model of chronic constriction injury of the infraorbital nerve].

Trigeminal neuralgia is a paroxysmal disorder with severely disabling facial pain and thus continues to be a real therapeutic challenge. At present there are few effective drugs for treatment of this pain. The present study was aimed to explore the involvement of BK(Ca) channels and Kv channels in the mechanical allodynia in a rat model of trigeminal neuropathic pain. Here the effectiveness of drug target injection at the trigeminal ganglion through the infraorbital foramen was first evaluated by immunofluorescence and animal behavior test. Trigeminal neuropathic pain model was established by chronic constriction injury of the infraorbital nerve (ION-CCI) in rats. BK(Ca) channel agonist and Kv channel antagonist were administered into the trigeminal ganglion in ION-CCI rats and sham rats by the above target injection method, and the facial mechanical pain threshold was measured. The results showed that the drug could accurately reach the trigeminal ganglion by target injection which was more effective than that by the normal injection around infraorbital foramen. Rats suffered significant mechanical allodynia in the whisker pad of the operated side from 6 d to 42 d after ION-CCI. BK(Ca) channel agonist NS1619 significantly and dose-dependently attenuated the facial mechanical allodynia and increased the facial mechanical pain threshold in ION-CCI rats 15 d after operation. Kv antagonist 4-AP was able to reduce the threshold in ION-CCI rats when facial mechanical threshold was partly recovered and relatively stable on the 35th day after operation. These results suggest that BK(Ca) channel agonist NS1619 and Kv channel antagonist 4-AP can significantly affect the rats' facial mechanical pain threshold after ION-CCI. Activation of BK(Ca) channels may be related to the depression of the primary afferent neurons in trigeminal neuropathic pain pathways. Activation of Kv channels may exert a tonic inhibition on the trigeminal neuropathic pain.

Molecular dissection of I(A) in cortical pyramidal neurons reveals three distinct components encoded by Kv4.2, Kv4.3, and Kv1.4 alpha-subunits.

The rapidly activating and inactivating voltage-gated K(+) (Kv) current, I(A), is broadly expressed in neurons and is a key regulator of action potential repolarization, repetitive firing, backpropagation (into dendrites) of action potentials, and responses to synaptic inputs. Interestingly, results from previous studies on a number of neuronal cell types, including hippocampal, cortical, and spinal neurons, suggest that macroscopic I(A) is composed of multiple components and that each component is likely encoded by distinct Kv channel alpha-subunits. The goals of the experiments presented here were to test this hypothesis and to determine the molecular identities of the Kv channel alpha-subunits that generate I(A) in cortical pyramidal neurons. Combining genetic disruption of individual Kv alpha-subunit genes with pharmacological approaches to block Kv currents selectively, the experiments here revealed that Kv1.4, Kv4.2, and Kv4.3 alpha-subunits encode distinct components of I(A) that together underlie the macroscopic I(A) in mouse (male and female) cortical pyramidal neurons. Recordings from neurons lacking both Kv4.2 and Kv4.3 (Kv4.2(-/-)/Kv4.3(-/-)) revealed that, although Kv1.4 encodes a minor component of I(A), the Kv1.4-encoded current was found in all the Kv4.2(-/-)/Kv4.3(-/-) cortical pyramidal neurons examined. Of the cortical pyramidal neurons lacking both Kv4.2 and Kv1.4, 90% expressed a Kv4.3-encoded I(A) larger in amplitude than the Kv1.4-encoded component. The experimental findings also demonstrate that the targeted deletion of the individual Kv alpha-subunits encoding components of I(A) results in electrical remodeling that is Kv alpha-subunit specific.

Regulation of antiarrhythmic drug propafenone effects on the c-type Kv1.4 potassium channel by PHo and K+.

The effects of the antiarrhythmic drug propafenone at c-type kv1.4 channels in Xenopus laevis oocytes were studied with the two-electrode voltage-clamp technique. Defolliculated oocytes (stage V-VI) were injected with transcribed cRNAs of ferret Kv1.4 Delta N channels. During recording, oocytes were continuously perfused with control solution or propafenone. Propafenone decreased the currents during voltage steps. The block was voltage-, use-, and concentration- dependent manners. The block was increased with positive going potentials. The voltage dependence of block could be fitted with the sum of monoexponential and a linear function. Propafenone accelerated the inactivate of current during the voltage step. The concentration of half-maximal block (IC(50)) was 121 microM/L. With high, normal, and low extracellular potassium concentrations, the changes of IC(50) value had no significant statistical differences. The block of propafenone was PH- dependent in high-, normal- and low- extracellular potassium concentrations. Acidification of the extracellular solution to PH 6.0 increased the IC(50) values to 463 microM/L, alkalization to PH 8.0 reduced it to 58 microM/L. The results suggest that propafenone blocks the Kv1.4 Delta N channel in the open state and give some hints for an intracellular site of action.

Ginsenoside Rg3 inhibits human Kv1.4 channel currents by interacting with the Lys531 residue.

We have demonstrated previously that the 20(S) but not the 20(R) form of ginsenoside Rg(3) inhibited K(+) currents flowing through Kv1.4 (hKv1.4) channels expressed in Xenopus laevis oocytes, pointing to the presence of specific interaction site(s) for Rg(3) in the hKv1.4 channel. In the current study, we sought to identify this site(s). To this end, we first assessed how point mutations of various amino acid residues of the hKv1.4 channel affected inhibition by 20(S)-ginsenoside Rg(3) (Rg(3)). Lys531 residue is known to be a key site for K(+) activation and to be part of the extracellular tetraethylammonium (TEA) binding site; the mutation K531Y abolished the Rg(3) effect and made the Kv1.4 channel sensitive to TEA applied to the extracellular side of the membrane. Mutations of many other residues, including the pH sensitive-site (H507Q), were without any significant effect. We next examined whether K(+) and TEA could alter the effect of Rg(3) and vice versa. We found that 1) raising [K(+)](o) reduced the inhibitory effect of Rg(3) on hKv1.4 channel currents, whereas Rg(3) shifted the K(+) activation curve to the right, and 2) TEA caused a rightward shift of the Rg(3) concentration-response curve of wild-type hKv1.4 channel currents, whereas Rg(3) caused a rightward shift of the TEA concentration-response curve of K531Y mutant channel currents. The docked modeling revealed that Lys531 plays a key role in forming hydrogen bonds between Rg(3) and hKv1.4 channels. These results indicate that Rg(3) inhibits the hKv1.4 channel current by interacting with residue Lys531.

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4.

Voltage-gated K(+) channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K(+) currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At -60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K(+) channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K(+) channels.

Curcumin potently blocks Kv1.4 potassium channels.

Curcumin, a major constituent of the spice turmeric, is a nutriceutical compound reported to possess therapeutic properties against a variety of diseases ranging from cancer to cystic fibrosis. In whole-cell patch-clamp experiments on bovine adrenal zona fasciculata (AZF) cells, curcumin reversibly inhibited the Kv1.4K+ current with an IC50 of 4.4 microM and a Hill coefficient of 2.32. Inhibition by curcumin was significantly enhanced by repeated depolarization; however, this agent did not alter the voltage-dependence of steady-state inactivation. Kv1.4 is the first voltage-gated ion channel demonstrated to be inhibited by curcumin. Furthermore, these results identify curcumin as one of the most potent antagonists of these K+ channels identified thus far. It remains to be seen whether any of the therapeutic actions of curcumin might originate with its ability to inhibit Kv1.4 or other voltage-gated K+ channel.