Probing Binding Sites and Mechanisms of Action of an IKs Activator by Computations and Experiments

Yu Xu,Yuhong Wang,Mei Zhang,Min Jiang,Avia Rosenhouse-Dantsker,Tsjerk Wassenaar,Gea-Ny Tseng

doi:10.1016/j.bpj.2014.10.059

Abstract

The slow delayed rectifier (IKs) channel is composed of the KCNQ1 channel and KCNE1 auxiliary subunit, and functions to repolarize action potentials in the human heart. IKs activators may provide therapeutic efficacy for treating long QT syndromes. Here, we show that a new KCNQ1 activator, ML277, can enhance IKs amplitude in adult guinea pig and canine ventricular myocytes. We probe its binding site and mechanism of action by computational analysis based on our recently reported KCNQ1 and KCNQ1/KCNE1 3D models, followed by experimental validation. Results from a pocket analysis and docking exercise suggest that ML277 binds to a side pocket in KCNQ1 and the KCNE1-free side pocket of KCNQ1/KCNE1. Molecular-dynamics (MD) simulations based on the most favorable channel/ML277 docking configurations reveal a well-defined ML277 binding space surrounded by the S2-S3 loop and S4-S5 helix on the intracellular side, and by S4–S6 transmembrane helices on the lateral sides. A detailed analysis of MD trajectories suggests two mechanisms of ML277 action. First, ML277 restricts the conformational dynamics of the KCNQ1 pore, optimizing K+ ion coordination in the selectivity filter and increasing current amplitudes. Second, ML277 binding induces global motions in the channel, including regions critical for KCNQ1 gating transitions. We conclude that ML277 activates IKs by binding to an intersubunit space and allosterically influencing pore conductance and gating transitions. KCNE1 association protects KCNQ1 from an arrhythmogenic (constitutive current-inducing) effect of ML277, but does not preclude its current-enhancing effect.

Highlights

The slow delayed rectifier (IKs) channel is composed of two main components: the KCNQ1 channel and the KCNE1 auxiliary subunit [1]
Several Q1 activators are available, but they are limited by a lack of specificity (zinc pyrithione (ZnPy) activates Q1 and other members of the KCNQ family, which are important in regulating neuronal excitability [4]) or low potency (phenylboronic acid increases recombinant IKs with an EC50 of 1.6 mM [6])
We tested the effect of ML277 (ML, 1 mM) on native IKs of adult guinea pig and canine ventricular myocytes (Fig. 1, A–C, left panels, and Fig. S1 A)

Summary

Introduction

The slow delayed rectifier (IKs) channel is composed of two main components: the KCNQ1 channel and the KCNE1 auxiliary subunit [1] (abbreviated as Q1 and E1, respectively). IKs functions as a repolarization reserve in the human heart, i.e., it helps to limit action potential durations (APDs) under stressful conditions, when the b-adrenergic tone is high or when other repolarizing currents are suppressed [2,3]. There has been an increasing interest in developing Kv channel activators as therapeutic agents [4,5]. Several Q1 activators are available, but they are limited by a lack of specificity (zinc pyrithione (ZnPy) activates Q1 and other members of the KCNQ family, which are important in regulating neuronal excitability [4]) or low potency (phenylboronic acid increases recombinant IKs with an EC50 of 1.6 mM [6]). ML is selective for Q1 (R100-fold more potent in activating Q1 than KCNQ2 or KCNQ4) and highly potent (EC50 < 1 mM) [7]

Methods

Results

Conclusion