Stable Interaction between Voltage-Activated Ca2+ Channel α1 and β Subunits Revealed by Translocatable β Systems

Abstract

High-voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit and two auxiliary α2δ and β subunits. Although it is well established that CaV β promotes cell surface expression and regulates the gating properties of CaV channels, the stability of the α1-β interaction in vivo remains unclear. Here, we address this issue by engineering translocatable CaV β systems that allow the real-time measurement of the coupling in live imaging and patch-clamp. In cells without CaV α1B expression, all constructed β subunits except palmitoylated β2a were translocated to the intracellular target membranes by rapamycin application. However, in cells co-expressed with CaV α1B no translocation of the β subunits was measured up to 2 hrs. In addition, rapamycin-induced recruitment of CaV β subunits to the plasma membrane did not affected the gating properties of CaV channels. In contrast, double mutation of CaV β subunits was shown to be dissociated easily from CaV α1 by rapamycin application. In these mutant forms, dissociation of β subunit from CaV α1B lead to the decrease in current amplitude, PIP2 sensitivity and current recovery from Gβγ inhibition. Furthermore, it inhibited inactivation and shifted the voltage dependent IV curve to the right in the live cells. When cells were cotransfected with double mutated CaV β1b and β2a together, rapamycin lead to dissociation of β1b, but not β2a, from CaV α1B. The CaV α1B separated from β1b did not further interact with CaV β2a, suggesting that once dissociated, CaV α1 do not interact with other β. Taken together, our data demonstrate that the interaction of CaV α1 with β subunit is very stable in live cells.