Johann Kiehn

Molecular Determinants of Dofetilide Block of HERG K+ 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 (IKr). HERG/IKr channels are a prime target for the pharmacological management of arrhythmias and are selectively blocked by class III antiarrhythmic methanesulfonanilide drugs, such as dofetilide, E4031, and MK-499, at submicromolar concentrations. By contrast, the closely related bovine ether-a-go-go channel (BEAG) is 100-fold less sensitive to dofetilide. To identify the molecular determinants for dofetilide block, we first engineered chimeras between HERG and BEAG and then used site-directed mutagenesis to localize single amino acid residues responsible for block. Using constructs heterologously expressed in Xenopus oocytes, we found that transplantation of the S5-S6 linker from BEAG into HERG removed high-affinity block by dofetilide. A point mutation in the S5-S6 linker region, HERG S620T, abolished high-affinity block and interfered with C-type inactivation. Thus, our results indicate that important determinants of dofetilide binding are localized to the pore region of HERG. Since the loss of high-affinity drug binding was always correlated with a loss of C-type inactivation, it is possible that the changes observed in drug binding are due to indirect allosteric modifications in the structure of the channel protein and not to the direct interaction of dofetilide with the respective mutated site chains. However, the chimeric approach was not able to identify domains outside the S5-S6 linker region of the HERG channel as putative candidates involved in drug binding. Moreover, the reverse mutation BEAG T432S increased the affinity of BEAG K+ channels for dofetilide, whereas C-type inactivation could not be recovered. Thus, the serine in position HERG 620 may participate directly in dofetilide binding; however, an intact C-type inactivation process seems to be crucial for high-affinity drug binding.

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 inXenopus 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 μm), 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 μm Ro-20–1724 was applied to the HERG 4mchannel, 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 μm, 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 (IKr). 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 µM 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.

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.

HERG Potassium Channel Activation Is Shifted by Phorbol Esters via Protein Kinase A-dependent Pathways

We investigated the effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on the rapid component of the delayed rectifier potassium current, I Kr, in guinea pig cardiomyocytes and found that theI Kr current amplitude was reduced by 20% with 10 nm PMA and 44% with 100 nm PMA. The ether-a-go-go-related gene (HERG) encodesI Kr in human heart. We expressedHERG heterologously in Xenopus oocytes and investigated the effects of PMA on the delayed rectifier potassium current. Upon application of PMA in a concentration of 100 nm, we found a similar reduction of HERG outward current amplitude by 59%. This reduction was due to a shift in the HERG activation curve by 37 mV. The ED50 for the PMA-induced shift was 9.0 nm. The inactive 4α-phorbol 12-myristate 13-acetate (4α-PMA) had no effect. PMA is known to act by stimulating distinct protein kinase cascades. Additional application of the specific protein kinase C inhibitors chelerythrine (10 μm) or bisindolylmaleimide (1 μm) could not attenuate the PMA-induced shift. In contrast, the shift by PMA was reduced significantly when the specific protein kinase A (PKA) inhibitors H89 (50 μm) or KT5720 (2.5 μm) were applied. Forskolin (400 μm), an activator of the adenylate cyclase that results in PKA activation, shifted the HERG activation curve by 14 mV. Moreover the specific protein kinase C activator 1-stearoyl-2-arachidonylglycerol (10 μm) showed no effect. Our data suggest that mainly PKA is mediating the shift of the HERG activation kinetics.

Comparison of Binding and Block Produced by Alternatively Spliced Kvβ1 Subunits

Voltage-gated K+ (Kv) channels consist of α subunits complexed with cytoplasmic Kvβ subunits. Kvβ1 subunits enhance the inactivation of currents expressed by the Kv1 α subunit subfamily. Binding has been demonstrated between the C terminus of Kvβ1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker α subunits. Here we have examined the interaction and functional properties of two alternatively spliced human Kvβ subunits, 1.2 and 1.3, with Kvα subunits 1.1, 1.2, 1.4, and 1.5. In the yeast two-hybrid assay, we found that both Kvβ subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvα subunit. In functional experiments, we found differences in modulation of Kv1α subunit currents that we attribute to the unique N-terminal domains of the two Kvβ subunits. Both Kvβ subunits act as open channel blockers at physiological membrane potentials, but hKvβ1.2 is a more potent blocker than hKvβ1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1.5. Moreover, hKvβ1.2 is sensitive to redox conditions, whereas hKvβ1.3 is not. We suggest that different Kvβ subunits extend the range over which distinct Kv1α subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions.

Adrenergic regulation of the rapid component of the cardiac delayed rectifier potassium current, IKr, and the underlying hERG ion channel

Abstract.Ventricular arrhythmias are often precipitated by physical or emotional stress, in particular in patients with ischemic heart disease or hereditary long QT syndrome. Stimulation of the sympathetic nervous system in response to exercise or emotional stress causes activation of cardiac α- and β-adrenoceptors. The rapid component of the delayed rectifier potassium current, IKr, and the underlying hERG potassium channel are critical for the regulation of heart rhythm. Recent experimental studies revealed that hERG/IKr currents are modulated by α- and β-adrenergic stimulation, providing a pathophysiological explanation for the increased incidence of arrhythmias during stress. This review summarizes the current knowledge on hERG/IKr channel modulation by adrenergic activity. In addition, therapeutic approaches to future effective, more genotype-specific antiarrhythmic therapies are discussed.

Regulation of HERG potassium channel activation by protein kinase C independent of direct phosphorylation of the channel protein

OBJECTIVE Patients with HERG-associated long QT syndrome typically develop tachyarrhythmias during physical or emotional stress. Previous studies have revealed that activation of the beta-adrenergic system and consecutive elevation of the intracellular cAMP concentration regulate HERG channels via protein kinase A-mediated phosphorylation of the channel protein and via direct interaction with the cAMP binding site of HERG. In contrast, the influence of the alpha-adrenergic signal transduction cascade on HERG currents as suggested by recent reports is less well understood. The aim of the present study was to elucidate the biochemical pathways of the protein kinase C (PKC)-dependent regulation of HERG currents. METHODS HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. RESULTS Application of the phorbol ester PMA, an unspecific protein kinase activator, shifted the voltage dependence of HERG activation towards more positive potentials. This effect could be mimicked by activation of conventional PKC isoforms with thymeleatoxin. Coexpression of HERG with the beta-subunits minK or hMiRP1 did not alter the effect of PMA. Specific inhibition of PKC abolished the PMA-induced activation shift, suggesting that PKC is required within the regulatory mechanism. The PMA-induced effect could still be observed when the PKC-dependent phosphorylation sites in HERG were deleted by mutagenesis. Cytoskeletal proteins such as actin filaments or microtubules did not affect the HERG activation shift. CONCLUSION In addition to the known effects of PKA and cAMP, HERG channels are also modulated by PKC. The molecular mechanisms of this PKC-dependent process are not completely understood but do not depend on direct PKC-dependent phosphorylation of the channel.

QTc Prolongation by Grapefruit Juice and Its Potential Pharmacological Basis HERG Channel Blockade by Flavonoids

Background—A high intake of dietary flavonoids, which are abundant in fruits, vegetables, tea, and wine, is known to reduce cardiovascular mortality. The effects of flavonoids on cardiac electrophysiology, which theoretically may have both antiarrhythmic and proarrhythmic consequences, have not been studied systematically to date. Methods and Results—We screened a broad spectrum of flavonoids for their inhibitory activity on HERG channels by using heterologous expression in Xenopus oocytes. At a concentration of 1 mmol/L, 10 compounds caused a significant inhibition of HERG currents, whereas 11 other flavonoids had no effect. The IC50 value for HERG block by naringenin, the most potent inhibitor, was 102.3 &mgr;mol/L in Xenopus oocytes and 36.5 &mgr;mol/L in HEK cells. To demonstrate the physiological relevance of these findings, we studied the effects of pink grapefruit juice, which contains large amounts of naringenin glycosides (>1000 &mgr;mol/L), in human volunteers. In 10 persons, we observed a peak QTc prolongation of 12.5±4.2 ms 5 hours after oral ingestion of 1 L of grapefruit juice. This effect was significant (P=0.02). Conclusions—We found a significant QTc prolongation by grapefruit juice in healthy volunteers, probably caused by block of HERG channels by flavonoids. These findings reveal new perspectives on the potential for dietary modification of cardiac electrophysiology.

Human Cardiac Inwardly-Rectifying K+ Channel Kir2.1b Is Inhibited by Direct Protein Kinase C-Dependent Regulation in Human Isolated Cardiomyocytes and in an Expression System

Background—Protein kinases A (PKA) and C (PKC) are activated in ischemic preconditioning and heart failure, conditions in which patients develop arrhythmias. The native inward rectifier potassium current (IK1) plays a central role in the stabilization of the resting membrane potential and the process of arrhythmogenesis. This study investigates the functional relationship between PKC and IK1. Methods and Results—In whole-cell patch-clamp experiments with isolated human atrial cardiomyocytes, the IK1 was reduced by 41% when the nonspecific activator of PKC phorbol 12 myristate 13-acetate (PMA; 100 nmol/L) was applied. To investigate the effects of PKC on cloned channel underlying parts of the native IK1, we expressed Kir2.1b heterologously in Xenopus oocytes and measured currents with the double-electrode voltage-clamp technique. PMA decreased the current by an average of 68%, with an IC50 of 0.68 nmol/L. The inactive compound 4-&agr;-PMA was ineffective. Thymeleatoxin and 1-oleolyl-2-acetyl-sn-glycerol, 2 specific activators of PKC, produced effects similar to those of PMA. Inhibitors of PKC, ie, staurosporine and chelerytrine, could inhibit the PMA effect (1 nmol/L) significantly. After mutation of the PKC phosphorylation sites (especially S64A and T353A), PMA became ineffective. Conclusions—The human IK1 in atrial cardiomyocytes and one of its underlying ion channels, the Kir2.1b channel, is inhibited by PKC-dependent signal transduction pathways, possibly contributing to arrhythmogenesis in patients with structural heart disease in which PKC is activated.