Examining the Role of Phosphorylation on Interactions between the Cardiac Potassium Channel Alpha-Subunits hERG and KVLQT1

Abstract

KvLQT1 and hERG are the voltage-gated K+ channel α-subunits of the channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and cell culture have motivated our investigations into interactions between hERG and KvLQT1. These data suggest that a dynamic physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which HERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is thought to play a regulatory role in this process: modifying the phosphorylation state of each the proteins has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP concentration. Through classic biochemical assays and quantitative FRET approaches, we aim to characterize the effects of phosphorylation in regulating interactions between KvLQT1 and hERG in cellular model systems. We have developed ion channel fusions to fluorescent proteins, which include hERG and KvLQT1 phosphonull and phosphomimetic mutants. We hypothesize that phosphorylation abrogates protein-protein interactions, as suggested by findings that increased cAMP levels leads to decreased hERG-KvLQT1 interaction. This work potentially furthers our understanding of hERG-KvLQT1 interactions and may elucidate mechanisms that underlie many types of arrhythmia as well as characterize novel interactions between two distinct potassium channel families.