Abstract

Initiation of skeletal muscle contraction is triggered by rapid activation of RyR1 channels in response to sarcolemmal depolarization. RyR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of CaV1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both CaV1.1 and RyR1 activations, reported as fluorescence changes. We discovered that each of the four human CaV1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in CaV1.1 activation; VSD-II, VSD-III and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca2+ release. The prominent role of VSD-I in governing CaV1.1 activation was also confirmed using a naturally-occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished CaV1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally-relevant allosteric model of CaV activation, which accounted for both time- and voltage-dependent properties of CaV1.1, to predict VSD-pore coupling energies (W), we found that VSD-I contributed the most energy (W1> 3kT) towards the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (W< 1kT). This study settles the long-standing question of how CaV1.1, a slowly-activating channel, can trigger RyR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human CaV1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels