TASK1 (K(2P)3.1) K(+) channel inhibition by endothelin-1 is mediated through Rho kinase-dependent phosphorylation.

BACKGROUND AND PURPOSE: TASK1 (K(2P)3.1) two-pore-domain K(+) channels contribute substantially to the resting membrane potential in human pulmonary artery smooth muscle cells (hPASMC), modulating vascular tone and diameter. The endothelin-1 (ET-1) pathway mediates vasoconstriction and is an established target of pulmonary arterial hypertension (PAH) therapy. ET-1-mediated inhibition of TASK1 currents in hPASMC is implicated in the pathophysiology of PAH. This study was designed to elucidate molecular mechanisms underlying inhibition of TASK1 channels by ET-1. EXPERIMENTAL APPROACH: Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record TASK1 currents from hPASMC and Xenopus oocytes. KEY RESULTS: ET-1 inhibited TASK1-mediated I(KN) currents in hPASMC, an effect attenuated by Rho kinase inhibition with Y-27632. In Xenopus oocytes, TASK1 current reduction by ET-1 was mediated by endothelin receptors ET(A) (IC(50) = 0.08 nM) and ET(B) (IC(50) = 0.23 nM) via Rho kinase signalling. TASK1 channels contain two putative Rho kinase phosphorylation sites, Ser(336) and Ser(393) . Mutation of Ser(393) rendered TASK1 channels insensitive to ET(A) - or ET(B)-mediated current inhibition. In contrast, removal of Ser(336) selectively attenuated ET(A) -dependent TASK1 regulation without affecting the ET(B) pathway. CONCLUSIONS AND IMPLICATIONS: ET-1 regulated vascular TASK1 currents through ET(A) and ET(B) receptors mediated by downstream activation of Rho kinase and direct channel phosphorylation. The Rho kinase pathway in PASMC may provide a more specific therapeutic target in pulmonary arterial hypertension treatment.

Central role of PKCα in isoenzyme-selective regulation of cardiac transient outward current Ito and Kv4.3 channels.

The transient outward current I(to) is an important determinant of the early repolarization phase. I(to) and its molecular basis Kv4.3 are regulated by adrenergic pathways including protein kinase C. However, the exact regulatory mechanisms have not been analyzed yet. We here analyzed isoenzyme specific regulation of Kv4.3 and I(to) by PKC. Kv4.3 channels were expressed in Xenopus oocytes and currents were measured with double electrode voltage clamp technique. Patch clamp experiments were performed in isolated rat cardiomyocytes. Unspecific PKC stimulation with PMA resulted in a reduction of Kv4.3 current. Similar effects could be observed after activation of conventional PKC isoforms by TMX. Both effects were reversible by pharmacological inhibition of the conventional PKC isoenzymes (Gö6976). In contrast, activation of the novel PKC isoforms (ingenol) did not significantly affect Kv4.3 current. Whereas TMX-induced PKC activation was not attenuated inhibition of PKCß, inhibition of PKCα with HBDDE prevented inhibitory effects of both PMA and TMX. Accordingly, stimulatory effects of PMA and TMX could be mimicked by the α-isoenzyme selective PKC activator iripallidal. Further evidence for the central role of PKCα was provided with the use of siRNAs. We found that PKCα siRNA but not PKCß siRNA abolished the TMX induced effect. In isolated rat cardiomyocytes, PMA dependent I(to) reduction could be completely abolished by pharmacologic inhibition of PKCα. In summary we show that PKCα plays a central role in protein kinase C dependent regulation of Kv4.3 current and native I(to). These results add to the current understanding of isoenzyme selective ion channel regulation by protein kinases.

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein.

BACKGROUND AND PURPOSE: Two-pore-domain potassium (K2P) channels mediate potassium background (or 'leak') currents, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Inhibition of K2P currents permits membrane potential depolarization and excitation. As expected for key regulators of excitability, leak channels are under tight control from a plethora of stimuli. Recently, signalling via protein tyrosine kinases (TKs) has been implicated in ion channel modulation. The objective of this study was to investigate TK regulation of K2P channels. EXPERIMENTAL APPROACH: The two-electrode voltage clamp technique was used to record K2P currents in Xenopus oocytes. In addition, K2P channels were studied in Chinese hamster ovary (CHO) cells using the whole-cell patch clamp technique. KEY RESULTS: Here, we report inhibition of human K2P3.1 (TASK-1) currents by the TK antagonist, genistein, in Xenopus oocytes (IC50=10.7 microM) and in CHO cells (IC50=12.3 microM). The underlying molecular mechanism was studied in detail. hK2P3.1 was not affected by genistin, an inactive analogue of genistein. Perorthovanadate, an inhibitor of tyrosine phosphatase activity, reduced the inhibitory effect of genistein. Current reduction was voltage independent and did not require channel protonation at position H98 or phosphorylation at the single TK phosphorylation site, Y323. Among functional hK2P family members, genistein also reduced K2P6.1 (TWIK-2), K2P9.1 (TASK-3) and K2P13.1 (THIK-1) currents, respectively. CONCLUSIONS AND IMPLICATIONS: Modulation of K2P channels by the TK inhibitor, genistein, represents a novel molecular mechanism to alter background K+ currents.

The cardiac hERG/IKr potassium channel as pharmacological target: structure, function, regulation, and clinical applications.

Human ether-a-go-go-related gene (hERG) potassium channels conduct the rapid component of the delayed rectifier potassium current, IKr, which is crucial for repolarization of cardiac action potentials. Moderate hERG blockade may produce a beneficial class III antiarrhythmic effect. In contrast, a reduction in hERG currents due to either genetic defects or adverse drug effects can lead to hereditary or acquired long QT syndromes characterized by action potential prolongation, lengthening of the QT interval on the surface ECG, and an increased risk for "torsade de pointes" arrhythmias and sudden death. This undesirable side effect of non-antiarrhythmic compounds has prompted the withdrawal of several blockbuster drugs from the market. Studies on mechanisms of hERG channel inhibition provide significant insights into the molecular factors that determine state-, voltage-, and use-dependency of hERG current block. In addition, crucial properties of the high-affinity drug binding site in hERG and its interaction with drug molecules have been identified, providing the basis for more refined approaches in drug design, safety pharmacology and in silico modeling. Recently, mutations in hERG have been shown to cause current increase and hereditary short QT syndrome with a high risk for life-threatening arrhythmias. Finally, the discovery of adrenergic mechanisms of hERG channel regulation as well as the development of strategies to enhance hERG currents and to modify intracellular hERG protein processing may provide novel antiarrhythmic options in repolarization disorders. In conclusion, the increasing understanding of hERG channel function and molecular mechanisms of hERG current regulation could improve prevention and treatment of hERG-associated cardiac repolarization disorders.

[Molecular basis of primary electrical heart diseases]. / Molekulare Grundlagen primär elektrischer Herzerkrankungen.

The last decade has seen rapid progress in our understanding of the molecular basis of arrhythmias, particularly concerning hereditary arrhythmia syndromes. This has led to significant improvement regarding differentiation, risk stratification and therapy in these patients and their families. However, there is mounting evidence that the knowledge obtained by studying these rare monogenic disorders will also enable us to dissect the molecular mechanisms underlying polygenetic and multi-factorial arrhythmias that are by far more common in clinical practice. The goal of this review is to give a brief overview of current knowledge on the molecular basis of primary electrical heart diseases. A focus is on the long QT syndrome.

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome.

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.

Antiarrhythmic drug carvedilol inhibits HERG potassium channels.

OBJECTIVE: The aryloxypropanolamine carvedilol is a multiple action cardiovascular drug with blocking effects on alpha-receptors, beta-receptors, Ca(2+)-channels, Na(+)-channels and various native cardiac K(+) channels, thereby prolonging the cardiac action potential. In a number of clinical trials with patients suffering from congestive heart failure, carvedilol appeared to be superior to other beta-blocking agents in reducing total mortality. Given the multiple pharmacological actions of carvedilol, this may be due to specific channel blockade rather than beta-antagonistic activity. Since human ether-a-go-go related gene (HERG) K(+)channels play a critical role in the pathogenesis of cardiac arrhythmias and sudden cardiac death, the effects of carvedilol on HERG K(+)channels were investigated. METHODS: Double-electrode voltage-clamp experiments were performed on HERG potassium channels which were expressed heterologously in Xenopus oocytes. RESULTS: Carvedilol at a concentration of 10 microM blocked HERG potassium tail currents by 47%. The electrophysiological characteristics of HERG, i.e. activation, steady-state inactivation and recovery from inactivation were not affected by carvedilol. Inhibition of current gradually increased from 0% immediately after the test pulse to about 80% at 600 ms with subsequent marginal changes of current kinetics during the resting 29 s, indicating a very fast open channel block by carvedilol as the major blocking mechanism. CONCLUSION: This is the first study demonstrating that carvedilol blocks HERG potassium channels. The biophysical data presented in this study with a potentially antiarrhythmic effect may contribute to the positive outcome of clinical trials with carvedilol.

Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A.

We investigated the role of protein kinase A (PKA) in regulation of the human ether-a-go-go-related gene (HERG) potassium channel activation. HERG clones with single mutations destroying one of four consensus PKA phosphorylation sites (S283A, S890A, T895A, S1137A), as well as one clone carrying all mutations with no PKA phosphorylation sites (HERG 4M) were constructed. These clones were expressed heterologously in Xenopus oocytes, and HERG potassium currents were measured with the two microelectrode voltage clamp technique. Application of the cAMP-specific phosphodiesterase (PDE IV) inhibitor Ro-20-1724 (100 microM), which results in an increased cAMP level and PKA stimulation, induced a reduction of HERG wild type outward currents by 19.1% due to a shift in the activation curve of 12.4 mV. When 100 microM Ro-20-1724 was applied to the HERG 4M channel, missing all PKA sites, there was no significant shift in the activation curve, and the current amplitude was not reduced. Furthermore, the adenylate cyclase activator forskolin that leads to PKA activation (400 microM, 60 min), shifted HERG wild type channel activation by 14.1 mV and reduced currents by 39.9%, whereas HERG 4M channels showed only a small shift of 4.3 mV and a weaker current reduction of 22.3%. We conclude that PKA regulates HERG channel activation, and direct phosphorylation of the HERG channel protein has a functional role that may be important in regulation of cardiac repolarization.

Inhibitory effects of the class III antiarrhythmic drug amiodarone on cloned HERG potassium channels.

The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac-delayed rectifier K+ current (I(Kr)). HERG channels are one primary target for the pharmacological management of arrhythmias. In this study, we investigated the acute effects of the class III antiarrhythmic drug amiodarone on HERG channels expressed heterologously in Xenopus oocytes by use of the two-microelectrode voltage clamp technique. Amiodarone blocked HERG channels with an IC50 of 9.8 microM with a maximum outward tail current reduction of 62.8%. The block consisted of two main components, a closed channel block that could not be reversed within the time of experiments and an open channel block with a slow unblock, having a recovery time constant of 73 s at -80 mV. Inactivation of the HERG channel at very positive potentials could not prevent amiodarone block. These results indicate that HERG channels can be blocked by amiodarone in closed, open and inactivated states. The block of open channels was cumulative, use-dependent and voltage-dependent. In summary, our data suggest that the strong class III antiarrhythmic action of amiodarone is at least partially based upon its acute inhibitory effects on HERG potassium channels.

A K+ single channel and whole-cell clamp study on the effects of levocromakalim in guinea pig portal vein cells.

Single channel cell-attached patch and whole-cell clamp experiments on the mode of action of the K+ channel opener (KCO), levcromakalim, were performed in guinea pig isolated portal vein cells. At +20 mV (135/23 mM K+ in bath/pipette), 10 microM levcromakalim activated K+ channels with a chord conductance of 23.2 pS (K(KCO)), which were sensitive to the blocker of ATP-dependent K+ channels (K(ATP)), glibenclamide. Voltage steps from -80 mV to +20 mV activated 4-aminopyridine-sensitive K+ channels of 6.5 pS with properties of delayed rectifier K+ channels (Kv). In patches which upon a previous voltage step had revealed the existence of Kv, levcromakalim reduced the open-probability of Kv, but it did not concomitantly activate K(KCO). During the course of the experiments, but unrelated to the presence of levcromakalim, large conductance K+ channels (BK(Ca)) appeared which could be inhibited by iberiotoxin, a selective blocker of BK(Ca), and by the membrane-permeant calcium buffer, BAPTA/AM, but not by glibenclamide. Whole-cell current-voltage (i-V) relations were established in response to voltage ramps from +50 mV to -100 mV; on subtraction of control i-V curves from i-V curves obtained in the presence of 10 microM levcromakalim, the KCO-induced K+ current remained which was proportional to voltage. This is not compatible with the upward-bent curvature predicted by the GHK current equation for purely resistive channels at high [K+]i versus low [K+]o. In conclusion, in the guinea pig portal vein cells, no evidence could be established for the hypotheses that KCOs may act via conversion of Kv to K(ATP) (Beech and Bolton 1989; Edwards et al. 1993) or by activation of BK(Ca) (Balwierczak et al. 1995). In these cells, mild inward rectification of the levcromakalim-induced current was observed which underlines their relationship to K(ATP) in other tissues.