A mutational and modeling study of a binding pocket for IKs activators between KCNQ1 and KCNE1.

Abstract

The IKs current formed by the co-assembly of KCNQ1 and KCNE1 plays an important role in cardiac repolarization. Loss-of-function mutations in KCNQ1 and KCNE1 are linked to LQT syndrome. Mefenamic acid (Mef) an NSAID, and DIDS a stilbene, are known to enhance IKs currents. The present study explores in detail the role of previously identified K41-KCNE1 in Mef action as well as uncovering the KCNQ1/KCNE1 binding site(s) of Mef and DIDS. In silico experiments of drug docking on a model of IKs channel and molecular dynamic (MD) simulations were used to map critical KCNQ1 and KCNE1 residues, followed by site-directed mutagenesis and functional analysis using whole cell patch clamp. Docking procedures and MD simulations show that Mef and DIDS bind between extracellular KCNE1 residues, specifically to the subunits of the external S6 helix; neighbouring S1; and the pore turret of the opposing subunit. Mef transforms the slowly activating WT IKs current (4:4 EQ) into a current with an almost linear waveform, which causes a left shift in activation V1/2 and inhibits tail current decay at −40 mV. These effects are prevented by the K41C-KCNE1 mutation in a stoichiometric manner. MD simulations and mutagenesis scanning identified three critical residues for Mef binding: W323-KCNQ1; Y46-KCNE1; and K41-KCNE1 in which Mef effects are greatly diminished. The effect of DIDS on WT-EQ are less pronounced than Mef, and K41C-KCNE1 was unable to fully prevent effects of DIDS. Our results show that both activators bind in the same general channel region but depend on different critical residues. These findings provide insights into a common drug-induced binding site, suggesting that the chemical structure of either compound could serve as a starting point for the development of IKs agonists.