β2A and β3 Differentially Modulate Time- and Voltage-Dependent Properties of Individual Voltage Sensors in the Human Cav1.2 Channel

Abstract

The human L-type calcium channel (CaV1.2) is a multi-protein complex consisting of the pore-forming α1c subunit and at least two auxiliary subunits, β and α2δ, which assemble in a 1:1:1 stoichiometry. β subunits regulate multiple aspects of CaV1.2 channel physiology, including surface expression, degradation and gating. Investigating the effect of β2a and β3 subunits on human CaV1.2 channels (α1c+α2δ) expressed in Xenopus oocytes, we found that the voltage dependences of total charge displacement (QV) and conductance (GV) were ∼10mV more negative in the presence of β2a. We tested the hypothesis that β2a and β3 subunits differently modulate the activation of the four voltage-sensor domains (VSDs) in the α1c subunit. Using the Voltage-Clamp Fluorometry technique, we fluorescently tracked the movement of individual VSD in conducting human CaV1.2 channels. We found that, in the presence of β2a, the voltage-dependent activation of VSDs II and III was left-shifted as compared to β3-associated CaV1.2 (VSD II with β2a: Vhalf= −31.1±1.0 mV, z= 4.5±0.8 e0; VSD II with β3: Vhalf= −26.5±1.8 mV, z= 2.4±0.1 e0; VSD III with β2a: Vhalf= −36.6±3.9 mV, z= 1.8±0.1 e0; VSD III with β3: Vhalf= −16.9±1.4 mV, z= 1.2±0.1 e0). Moreover, activation and deactivation kinetics of VSD II in channels associated with β2a were dramatically slower than in the presence of β3, while voltage- and time-dependent properties of VSD IV remained the same in the presence of either Cavβ subunit. These results suggest that, in spite of their highly-conserved sequence homology, different β subunit isoforms distinctly modulate the biophysical properties of CaV1.2 voltage sensors to mediate their effect on Ca conductance.