Structural Determinants of Voltage-Gated Calcium Channel Gating Properties

Abstract

Voltage-gated calcium channels (CaV) control most activity-dependent functions of excitable cells, but the molecular mechanism how they respond to membrane depolarization with specific gating properties remains unknown. CaV channels consist of four homologous but non-identical repeats (I, II, III, IV), each containing a separate voltage-sensing domain (VSD) arranged around the common channel pore. Within each VSD the positive gating charges (K0, R1-R4) in the transmembrane helix S4 sequentially interact with negative counter-charges in helices S2 and S3 to support the movement of the gating charges across the electrical field of the membrane and thus to activate or deactivate the channel. We combined MD simulation and Markov-state models with mutagenesis and electrophysiological analysis to study the structure, free energy levels, and transition times of the activated and resting states of two distinct voltage-sensing domains of the prototypical eukaryotic calcium channel CaV1.1. Structure models of CaV1.1 in the activated and resting states show that VSD I and VSD IV differ greatly regarding ion-pair interactions formed between gating charges and counter-charges in the outer negative cluster. These structural differences are consistent with the specific contributions of VSD I and IV to kinetics and voltage-dependence of CaV1.1 activation, respectively. The functional relevance of the predicted interactions was confirmed by site-directed mutagenesis and patch clamp analysis. The data show that ion-pairs which stabilize the activated state cause a left-shift of the voltage-dependence of activation in VSD I and IV. In contrast, ion-pairs stabilizing resting states in VSD I slow down transition times between states and thus determine slow activation kinetics of CaV1.1. Thus, MD simulations reliably predict functionally relevant intramolecular interactions and distinct thermodynamic properties of states and different kinetics of state transitions explain the specific contributions of VSD I and IV to gating properties of CaV1.1.