IKS Activators Shift their Binding Sites in the KCNQ1 Channel After KCNE1 Association - in Silico Predictions and Experimental Tests

Abstract

KCNQ1 associates with KCNE1 (Q1/E1) to form slow delayed rectifier (IKs) channels, a ‘repolarization reserve’ in human ventricles. IKs activators may be used to treat LQT syndromes. The level of E1 presence in Q1/E1 complexes can affect the potency of IKs activators and their therapeutic efficacy. We investigate the molecular mechanisms of ‘E1-induced change in potency’ for two experimental IKs activators: thimerosal (TMS) and ML277. TMS acts from outside the cell membrane and requires disulfide formation with native Cys to manifest its activator effect. TMS left shifts the half-maximum activation voltage (V0.5) of both Q1 and Q1/E1. In oocyte expression, TMS increases the estimated maximal current amplitude (Imax) of Q1/E1 but reduces Imax of Q1. ML277 acts from inside the cell membrane. ML277 left shifts V0.5 and increases Imax, with a higher potency in Q1 than Q1/E1. We identify two putative ‘drug-binding pockets’ in our experimentally validated 3-D models of Q1 and Q1/E1: one in the space between S3 and S4 helices accessible to the extracellular space and right above C214 on S3, and the other between S2-S3 and S4-S5 linkers accessible to the intracellular space. They are defined as putative TMS- and ML277-binding sites, respectively. In silico docking shows: (a) TMS salt-bridges with K218 when docked to Q1, but salt-bridges with R228 when docked to Q1/E1. (b) ML277 preferentially interacts with a ring of Arg on S2-S3 and S4-S5 linkers when docked to Q1, but aromatic stacks with a ring of Phe on S5 and S6 when docked to Q1/E1. Mutagenesis/voltage-clamp experiments are underway to test these predictions, and implications in drug potency are interpreted based on our knowledge of the structure-function relationship of IKs channel.