Modulation of HERG K+ Channels by Chronic Exposure to Activators and Inhibitors of PKA and PKC: Actions Independent of PKA and PKC Phosphorylation

Abstract

Background: Human ether-a-go-go-related gene (HERG) channel is the major molecular component of the native rapid delayed rectifier K⁺ current (I_Kr) that is a crucial determinant of cardiac repolarization. Impairment of I_Kr/HERG function is commonly believed to be a mechanism causing long QT syndromes (LQTS), a lethal ventricular tachyarrhythmia. The cAMP-dependent protein kinase A (PKA) and PKC activities are markedly increased in some pathological conditions of the heart such as heart failure. This study was designed to investigate the effects of acute and chronic exposure to PKA or PKC activators and inhibitors on HERG channel activities and to provide insight into the mechanisms for the modulations. Methods: Channel activity was measured in HEK293 cells stably expressing HERG using whole-cell patch-clamp techniques. Intracellular reactive oxygen species (ROS) were measured by CM-H2DFDA. Mitochondrial membrane potential (ΔΨ_m) was measured using JC-1 dye. HERG channel phosphorylation was assayed by [³²P]orthophosphate methods. Results: Acute exposure of cells to PKA or PKC activators by bath superfusion minimally affected I_HERG, and so did the PKA or PKC inhibitor. By comparison, prolonged exposure (chronic incubation) of cells to PKA or PKC activators significantly impaired HERG K⁺ channel function as reflected by reduced I_HERG density and positive shift of the steady-state activation curve. Antioxidants vitamin E and MnTBAP both abolished the depressive effects of PKA or PKC activators on HERG function. Further, both PKA and PKC activators stimulated production of intracellular reactive oxygen species (ROS), an effect efficiently prevented by antioxidants or by PKA and PKC inhibitors. Conclusions: HERG function is insensitive to PKA or PKC phosphorylation modulation per se, but can be impaired by the activators of PKA or PKC with long exposure likely via generation of ROS. In view of the critical role of HERG K⁺ channel in regulating cardiac repolarization and the sustained activation of both PKA and PKC in many pathological conditions of the heart such as heart failure, it is conceivable that HERG impairment by ROS accumulation induced by PKA and PKC contributes to the impaired cardiac repolarization.