Molecular Mapping of An IKS Channel Opener Reveals Crucial Interactions Between KCNE1 and the Kv7.1 Voltage Sensor Paddle

Abstract

Voltage-gated K+ channels co-assemble with accessory subunits to form macromolecular complexes. In heart, assembly of Kv7.1 pore-forming subunits with KCNE1 auxiliary subunits generates the repolarizing K+ current IKS. We and others, recently suggested a strategic location of KCNE1 wedged close to helices S1 and S4 of two adjacent Kv7.1 voltage sensing domains (VSD) and nearby helix S6 of another Kv7.1 subunit. Here we show that the IKS channel opener, diisothiocyanostilbene-2′,2′-disulfonic acid (DIDS) acts on IKS as a gating-modifier, thereby converting the time- and voltage-dependent channels into almost voltage- and time-independent currents. While DIDS activates Kv7.1, it does not affect Kv7.2. The two isothiocyanate functionalities are crucial for the potent activating effect of DIDS on IKS, since 4′-acetamido-4′-isothiocyanostilbene-2′,2′-disulfonic acid (SITS) that has only one of these groups and 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS), which lacks isothiocyanate groups and thus cannot form covalent bonds with amino acids, do not activate IKS currents. Mutagenesis and modeling data indicate that DIDS activates IKS by docking to an externally-accessible pocket, formed at the interface between the superficial N-terminal boundary of the KCNE1 transmembrane segment and the VSD paddle motif of Kv7.1. DIDS does not activate the channel complex formed by co-expression of KCNE1 and a chimeric Kv7.1 endowed with a Kv7.2 VSD paddle. DIDS binding at the Kv7.1 VSD-KCNE1 interface reveals that two lysine residues, K41 in KCNE1 and K218 in Kv7.1 S3-S4 linker are distant to about 10 [[Unable to Display Character: A]]. Thus, KCNE1 affects Kv7.1 channel gating by closely interacting with the VSD paddle motif.