KCNE3 Stabilizes the Voltage Sensor S4 of KCNQ1 Channel, KCNE1 Uncouples S4 and the Gate

Abstract

KCNEs are single-span transmembrane β-subunits that assemble with and modulate the biophysical properties of voltage-gated K+ (Kv) channels. In the heart, the pore forming α-subunit KCNQ1 associates with KCNE1 to form the slowly-activating, voltage-gated IKs channels that contribute to the repolarization of the cardiac action potential. In tissues such as the colon, stomach and kidney, KCNQ1 coassembles with the β-subunit KCNE3 to form voltage-independent K+ channels important for K+ and Cl- secretion. KCNE3 has also been shown to be expressed in the human heart, although its physiological function remains unknown. Different mechanisms have been proposed to explain how different KCNE subunits alter KCNQ1 gating and permeation. For instance, KCNE3 is assumed to lock the voltage sensor (S4) of KCNQ1 channel in the activated state, resulting in a constitutively open channel. Here, we use voltage clamp fluorometry (VCF) to understand how KCNE3 affects the voltage sensor and the gate of KCNQ1 channel. We show that KCNE3, contrary to what was previously assumed, allows S4 movement in KCNQ1/KCNE3 channels. KCNE3 shifts the closing and S4 movement of KCNQ1 to extreme hyperpolarized potentials, such that at physiological voltage range (−80 mV to +40 mV), the channel is always open. By decoupling S4 and the gate, either by mutations or PIP2 depletion, we show that KCNE3 mainly affects S4 movement in KCNQ1. Two negatively charged residues in the N-terminus of KCNE3 (D54 and D55) are, at least partly, responsible for stabilizing S4 in an outward position, therefore stabilizing KCNQ1/KCNE3 channels in the activated open state. Further, we unitized a triple mutation of KCNE3, previously shown to convert KCNQ1/KCNE1 channel to a KCNQ1/KCNE1-like current, and observe a decoupling of S4 and the gate