Examining the Role of Direct cAMP-Binding Versus PKA-Mediated Effects on Interactions between the Cardiac Potassium Channel α-Subunit Proteins Herg and KvLQT1

Abstract

KvLQT1 and hERG are the voltage-gated K+ channel α-subunits of 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 have motivated our investigations into interactions between hERG and KvLQT1. Moreover, reports of interplay between hERG and KvLQT1 have been described both in the literature and anecdotally, and our recent work demonstrated that the interaction is mediated by the C-termini of both α-subunits and is modulated by cAMP. We hypothesize that direct binding of cAMP to the putative cyclic nucleotide binding domain (CNBD) in the hERG C-terminus abrogates hERG-KvLQT1 interactions, as assessed by FRET analyses. We have developed ion channel fusions to GFP variants, which include hERG CNBD mutants as well as phospho-null and phospho-mimetic mutants. cAMP levels are altered in heterologous cells expressing ion channel constructs through membrane-permeable cAMP analogs or forskolin along with IBMX. Through classic biochemical assays and quantitative FRET approaches, we aim to delineate the direct role of cAMP from that of downstream, PKA-mediated effects in regulating interactions between KvLQT1 and hERG in model systems. 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.