Abstract

High voltage-activated (HVA) calcium channels (CaV) 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 alpha 2 delta and CaV beta. A long-standing issue is how cytoplasmic CaV beta can shift the voltage dependence of channel opening without altering gating currents. Tracking the movement of individual VSDs by voltage-clamp fluorometry in human CaV1.2 revealed that only the VSD from the second repeat (VSD II) is perturbed by CaV beta 3 in a construct combining a fluorophore-tagged VSD II (S1623C) with a quenching tryptophan within 11 & Aring; in the PD of repeat III (E1141W). The final construct, S612C_E1141W, exhibited a biphasic voltage-dependent fluorescence whose negative phase was enhanced by CaV beta 3. 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 CaV beta-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.