FRET-Based Assessments of Interactions Between KCNQ1 and HERG Potassium Channel α-Subunits

Abstract

Voltage-gated potassium channels play a critical role in repolarizing cardiomyocytes, and abnormal repolarization leads to prolonged action potential durations (APDs), which manifest clinically as Long-QT (LQT) syndrome and can result in malignant arrhythmias and sudden cardiac death. LQT1 is due to mutations in KCNQ1 (KvlQT1) α-subunits which underlie the cardiac IKs current while LQT2 results from reductions in IKr which is carried by KCNH2 (HERG) channels. IKr and IKs appear interconnected in normal hearts and function together to maintain appropriate APDs. However, electrophysiological studiesin LQT1 and LQT2 transgenic rabbits as well as in stable cell lines demonstrated downregulation of the reciprocal repolarizing currents. Cells overexpressing pore mutants of KCNQ1, and therefore lacking IKs, show decreased IKr current while cells overexpressing HERG pore mutants, having reduced IKr, show downregulated IKs. Direct interactions between the C-termini of KCNQ1 and HERG have been shown by surface plasmon resonance assays (Ren et al., 2010, PMID: 20833965). To further examine the interaction between KCNQ1 and HERG we have developed and characterized a library of fluorescent protein fusion constructs to utilize in quantitative microscopy experiments. We hypothesize that functionally relevant, dynamic interactions between KCNQ1 and HERG occur within the plasma membrane. We will test this hypothesis with several FRET-based assays aimed at demonstrating measureable interactions between KCNQ1 and HERG and establishing a foundation to study the dynamics of such interactions.