Date

2017

Department or Program

Biological Chemistry

Primary Wellesley Thesis Advisor

Louise E.O. Darling

Additional Advisor(s)

John Cameron

Additional Advisor

Elizabeth Oakes

Abstract

KvLQT1 and hERG are the α-subunits of the voltage-gated K+ 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 protein 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. In addition, a putative hERG cyclic nucleotide binding domain was proposed to directly bind cAMP with possible functional consequences on channel kinetics and interaction. Through classic biochemical assays and quantitative FRET approaches, we aim to characterize the effects of cAMP and phosphorylation in regulating interactions between KvLQT1 and hERG in a cellular model system. FRET analysis of hERG cyclic nucleotide binding domain mutant interaction with KvLQT1 in the presence of increased intracellular cAMP suggests that a direct binding mechanism for cAMP is unlikely as regulator of hERG-KvLQT1 interaction. Furthermore, FRET and co-IP quantification of phosphonull and phosphomimetic hERG and KvLQT1 mutants indicate that unphosphorylated hERG does not interact with KvLQT1, suggesting that hERG phosphorylation is required for wild-type proteins to interact. Phosphorylation of KvLQT1 appears to be the driving factor abrogating hERG-KvLQT1 interaction. This work furthers our understanding of hERG-KvLQT1 interactions and may elucidate mechanisms that underlie many types of arrhythmias as well as characterize novel interactions between two distinct potassium channel families.

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