BPS2026 – VSD III as the key regulator of excitation-contraction coupling in CaV1.1

Ca V 1.1 is a calcium channel expressed on the membrane of skeletal muscle cells. Upon depolarization, Ca V 1.1 rapidly causes the conformationally coupled ryanodine receptor 1(RyR1), expressed on the sarcoplasmic reticulum, to open and release the calcium necessary for muscle contraction. This mechanism is called excitation contraction coupling (ECC). At higher depolarization, Ca V 1.1 elicits slowly activating and small inward calcium currents, with distinct activation properties from those of ECC. These functional differences arise form intrinsic properties of the four voltage sensing domains (VSDs) constituting Ca V 1.1. Each VSD comprises of four helices (S1-S4) linked by intra-extracellular loops. S4 presents five to six equally spaced positive gating charges interacting with negative countercharges in the surrounding helices. These transient ion-pair interactions facilitate and tune the S4 motion in response to depolarization. While VSD I and IV were indicated to control the current’s properties (kinetics and voltage dependence), without affecting ECC, two independent studies proposed either VSD II or III to activate ECC. Here, by combining site-directed mutagenesis with electrophysiological and fluorescent calcium recordings, we identified VSD III as the sole controller of ECC. We applied two independent mutation strategies to analogous residues in the four VSDs, resulting in differential modulations of the currents, whereas only mutations in VSD III also affected ECC. These data, combined with molecular dynamics simulations and Ca V 1.1 chimeric analysis, strongly support the notion that ECC is activated by a non-canonical conformational state of VSD III and distinct VSD III state transitions are necessary for the activation of the currents and ECC.