Which CaV1.1 Voltage Sensor(s) Activate RYR1?

Abstract

The skeletal muscle L-type Ca­V channel is composed by the pore-forming α1s (CaV1.1) in association with α2δ-1, β1a and γ1 subunits in 1:1:1:1 stoichiometry. This large protein complex makes physical contact with Ryanodine (RyR1) channel located in the sarcoplasmic reticulum (SR), conferring voltage-dependence to the Ca2+ release channel, thus coupling electrical excitation with contraction. CaV1.1 channels possess four homologous but non-identical voltage sensing domains (VSDs), which undergo voltage-dependent rearrangements: we propose that one VSD is a stronger candidate than all others to be the sensor responsible for RyR1 voltage-dependent activation. We reconstituted human CaV1.1 complexes (α1s, α2δ-1, β1a, γ1 and stac3) in Xenopus oocytes. We used the voltage clamp fluorometry technique to simultaneously record Ca2+ currents (2mM external Ca2+) and the structural rearrangements of the four individual VSDs. We found that the voltage-dependent activation curves of the four VSDs are spread from −150 to +80mV, all preceding pore opening, with VSD-I and VSD-IV operating at more hyperpolarized potentials (VSD-I Vhalf=-35.6±2.2mV, z=1.0±0.01e0, VSD-IV Vhalf=-64.9±2.7mV, z=0.74±0.02e0) than VSD-II and VSD-III (VSD-II Vhalf=7.2±2.8mV, z=1.6±0.1e0, VSD-III Vhalf=-23.9±3mV, z=1.6±0.04e0). Each VSD exhibited distinct time-dependent properties, with VSD-I and VSD-II displaying slow activation kinetics (VSD-I τ=39.8±2ms, VSD-II τ=132±16ms, at 20mV), while VSD-III and VSD-IV activated rapidly (VSD-III τ=1.8±0.2ms, VSD-IV τ=2±1ms, at 20mV). The operation of the four voltage sensors was then investigated under a physiologically-relevant action potential (AP) clamp (skeletal muscle AP). Within the short AP clamp (∼5ms), no clear structural rearrangements of the slowest sensors (VSD-I and VSD-II) could be observed, while VSD-III and VSD-IV were readily activated. Based on i) kinetics, ii) voltage-dependence, iii) proximity with intracellular II-III loop (known to be a critical element in EC coupling), VSD-III is a strong candidate as sensor conferring voltage-dependent properties to RyR1 and the SR Ca2+ release.