EPR Spectroscopic Study of the Voltage-Sensor Domain (VSD) of the Human KCNQ1 Potassium Ion Channel

Abstract

KCNQ1 is a voltage-gated potassium channel modulated by members of the KCNE protein family. KCNQ1 is essential to both the cardiac action potential that mediates heartbeat and K+ homeostasis in the inner ear. KCNQ1 dysfunction has been linked to multiple diseases, including various cardiac arrhythmias, congenital deafness, and type II diabetes mellitus. Human KCNQ1 is a 676-residue protein consisting of 100-residue N-terminal cytosolic domain, followed by an ∼260-residue channel domain containing six transmembrane (TMD) helices, and a 300-residue cytosolic C-terminus. The first four TMD helices (S1-S4) form the voltage-sensor domain (VSD) that is linked to the pore domain (helices S5 and S6). Roughly 40% of the >200 reported disease-related mutations in the KCNQ1 gene result in amino acid substitutions in the VSD, making structural and dynamic studies of this domain important in unraveling molecular mechanisms in human pathophysiology. Site-directed spin labeling EPR is a very powerful structural biology technique to study the structural and conformational dynamics of membrane proteins in membrane environment. In this study, we have successfully expressed the Cysteine substituted Q1-VSD in E. Coli, purified into detergent micelles, labeled with MTSL spin labels, and collected CW-EPR spectra. We are developing several advanced EPR spectroscopic techniques (such as double electron-electron resonance (DEER) and electron spin echo envelope modulation (ESEEM)) for probing the structural and dynamic properties of Q1-VSD in a lipid bilayer environment. This study will provide important mechanistic information on the Q1-VSD channel upon binding with KCNE1.