Differential Molecular Motions of KCNE Subunits in the IKs Channel Complex Detected by Substituted Cysteine-Accessibility Test and Disulfide-Trapping Experiments

Abstract

The IKs channel consists of a pore-forming KCNQ1 channel and auxiliary KCNE subunits: KCNE1 as the major determinant of IKs gating kinetics while KCNE2 as a regulator of IKs current amplitude. Knowledge about the structural basis of KCNQ1 modulation by these different KCNE subunits can help the design of KCNE-specific IKs modulators as antiarrhythmic agents. Recent studies showed that the extracellular-juxtamembranous region of KCNE1 could adopt highly flexible structures and make gating state-dependent contacts with the voltage-sensing domain (VSD) and pore-domain (PD) of KCNQ1. KCNQ1-KCNE1 interactions in this extracellular-juxtamembranous region are critical for the IKs channel function, as evidenced by congenital arrhythmia-associated mutations identified in this region. Whether KCNE2 can do the same is unclear. We systematically probe the functional role of the extracellular-juxtamembranous region of KCNE1 (positions 36-47) and KCNE2 (positions 39-50), using cysteine-scanning mutagenesis followed by cysteine-accessibility test and disulfide-trapping experiments. MTSET modification of KCNE1 40C-46C alters the voltage-dependence of IKs activation and, for 44C-46C, reduces the current amplitude. There is a gradient in MTSET modification rates, from fast-reacting (41C-42C) to slow-reacting (44C-46C), with 43C reporting state-dependent accessibility: fast-reacting in open-state & slow-reacting in closed-state. While extensive disulfide-bond partners are found between KCNE1 36C-43C and KCNQ1 144C-147C, no such disulfide-bond partners can be identified between the equivalent KCNE2 positions and KCNQ1 140C-148C. We propose that the KCNE transmembrane helices reside in the junction between VSDs and PD of the KCNQ1 channel, with a similar orientation (with respect to KCNQ1) and position (with respect to membrane bilayer). However, their extracellular-juxtamembranous regions can make differential contacts with KCNQ1, that contribute to their differential effects on the KCNQ1 channel function and provide an opportunity for the design of KCNE-specific IKs modulators.