Regulation of voltage-sensing structures of CaV1.2 calcium channel by the auxiliary β-Subunit (2025). De Giorgis, D.; Mellado, G.; Garate, J.; Neely, A.

High voltage–activated (HVA) calcium channels (Ca_V) have four homologous but nonidentical repeats encompassing a voltage-sensing domain (VSD) and a quarter of the pore domain (PD). HVA can be modulated by at least two accessory subunits α₂δ and Ca_Vβ. A long-standing issue is how cytoplasmic Ca_Vβ can shift the voltage dependence of channel opening without altering gating currents. Tracking the movement of individual VSDs by voltage-clamp fluorometry in human Ca_V1.2 revealed that only the VSD from the second repeat (VSD II) is perturbed by Ca_Vβ₃ in a construct combining a fluorophore-tagged VSD II (S1623C) with a quenching tryptophan within 11 Å in the PD of repeat III (E1141W). The final construct, S612C_E1141W, exhibited a biphasic voltage-dependent fluorescence whose negative phase was enhanced by Ca_Vβ₃. This behavior was well described by a kinetic model that includes three states for VSD II of which the intermediate state contributes the most to pore opening in a Ca_Vβ-dependent manner, and that open channels with VSD II in the intermediate state would yield the lowest fluorescence emissions. Molecular dynamics simulation correlates a structure with two translocated arginines with frequent fluorophore-W contact between VSD II and the pore of open channels.