A single Cavβ can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels

Voltage-dependent calcium-channel ss subunits (Ca-V ss) strongly modulate pore-forming alpha(1) subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P-0). Despite their central role, it is unclear whether binding of a single Ca-V ss, or multiple Ca-V ss s, to an alpha(1), subunit governs the two distinct functions. Conventional experiments utilizing coexpression of alpha(1) and Ca-V ss subunits have been unable to resolve the ambiguity due to difficulties in establishing their stoichiometry in functional channels. Here, we unambiguously establish a 1:1 stoichiometry by covalently linking Ca-V ss(2b) to the carboxyl terminus of alpha(1C) (Ca(V)1.2), creating alpha(1C)center dot ss(2b). Recombinant L-type channels reconstituted in HEK 293 cells with alpha(1C)center dot ss(2b) supported whole-cell currents to the same extent as channels reconstituted via coexpression of the individual subunits. Analysis of gating charge showed alpha(1C)center dot ss 2b fully restored channel trafficking to the plasma membrane. Co-transfecting Ca-V ss(2a) with alpha(1C)center dot ss(2b) had little further impact on function. To rule out the possibility that fused Ca-V ss(2b) was interacting in trans with neighbouring alpha(1) molecules, alpha(1C)center dot ss(2b) was cotransfected with alpha(1B) (Ca(V)2.2), and pharmacological block with nimodipine showed an absence of alpha(1B) trafficking. These results establish that association of a single Ca-V ss with a pore-forming alpha(1), subunit captures the functional essence of HVA calcium channels, and introduce alpha(1)-Ca-V ss fusion proteins as a powerful new tool to probe structure-function mechanisms.