Voltage-sensor gating charge interactions bimodally regulate voltage dependence and kinetics of calcium channel activation

Voltage-sensing domains (VSDs) are highly conserved protein modules that regulate the activation of voltage-gated ion channels. In response to membrane depolarization, positive gating charges in the S4 helix of VSDs move across the membrane electric field, which is focused at the hydrophobic constriction site (HCS) in the center of the VSD. This conformational change is translated into opening of the channel gate. Transient interactions of the gating charges with negatively charged countercharges in the adjacent helices are critical for catalyzing this state transition and for determining its voltage dependence and kinetics. However, the mechanism by which the sequential interactions between the multiple gating- and countercharges regulate these properties remains poorly understood. Here, we analyze the state transitions of the first VSD of CaV1.1 using MD simulation of the channel exposed to an electric field and site-directed mutagenesis of gating and countercharges to investigate the role of their interactions in determining the gating properties of CaV1.1. Alanine substitutions of gating charges differentially altered the kinetics or voltage dependence of activation, depending on whether they pass the HCS (R2 and R3) or not (K0, R1, and R4). Alanine substitutions of countercharges differentially altered kinetics and voltage dependence, depending on whether they facilitate the transfer of gating charges across the HCS (E100 and D126), and whether they stabilize the activated (E87, E90, and E140) or the resting state (E100, D126). Thus, our results reveal basic mechanistic principles by which variable interactions between gating charges and countercharges regulate the gating properties of voltage-gated calcium channels.