Inhibition of cloned HERG potassium channels by the antiestrogen tamoxifen.

Tamoxifen is a nonsteroidal antiestrogen that is commonly used in the treatment of breast cancer. Although antiestrogenic drugs are generally believed not to cause acquired long QT syndrome (LQTS), concerns have been raised by recent reports of QT interval prolongation associated with tamoxifen treatment. Since blockade of human ether-a-go-go-related gene (HERG) potassium channels is critical in the development of acquired LQTS, we investigated the effects of tamoxifen on cloned HERG potassium channels to determine the electrophysiological basis for the arrhythmogenic potential of this drug. HERG channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. Tamoxifen blocked HERG potassium channels with an IC(50) value of 45.3 microM. Inhibition required channel opening and unblocking occurred very slowly. Analysis of the voltage-dependence of block revealed loss of inhibition at positive membrane potentials, indicating that strong channel inactivation prevented block by tamoxifen. No marked changes in electrophysiological parameters such as voltage-dependence of activation or inactivation, or inactivation time constant could be observed, and block was not frequency-dependent. This study demonstrates that HERG potassium channels are blocked by the antiestrogenic drug tamoxifen. We conclude that HERG current inhibition might be an explanation for the QT interval prolongation associated with this drug.

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.

Human cardiac inwardly-rectifying K+ channel Kir(2.1b) 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 Kir(2.1b) 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-alpha-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 Kir(2.1b) channel, is inhibited by PKC-dependent signal transduction pathways, possibly contributing to arrhythmogenesis in patients with structural heart disease in which PKC is activated.

The antidepressant drug fluoxetine is an inhibitor of human ether-a-go-go-related gene (HERG) potassium channels.

Fluoxetine is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Although this group of antidepressant drugs is generally believed to cause fewer proarrhythmic side effects compared with tricyclic antidepressants, serious concerns have been raised by case reports of tachycardia and syncopes associated with fluoxetine treatment. To determine the electrophysiological basis for the arrhythmogenic potential of fluoxetine, we investigated the effects of this drug on cloned human ether-a-go-go-related gene (HERG) potassium channels heterologously expressed in Xenopus oocytes using the two-microelectrode voltage-clamp technique. We found that fluoxetine blocked HERG channels with an IC(50) value of 3.1 microM. Inhibition occurred fast to open channels with very slow unbinding kinetics. Analysis of the voltage dependence of block revealed loss of inhibition at membrane potentials greater than 40 mV, indicating that channel inactivation prevented block by fluoxetine. No pronounced changes in electrophysiological parameters such as voltage dependence of activation or inactivation, or inactivation time constant could be observed, and block was not frequency-dependent. This is the first study demonstrating that HERG potassium channels are blocked by the selective serotonin reuptake inhibitor fluoxetine. We conclude that HERG current inhibition might be an explanation for the arrhythmogenic side effects of this drug.