A single CaVbeta can reconstitute both trafficking and macroscopic conductance of voltage-dependent calcium channels.
Voltage-dependent calcium-channel beta subunits (Ca(V)beta) strongly modulate pore-forming alpha(1) subunits by trafficking channel complexes to the plasma membrane and enhancing channel open probability (P(o)). Despite their central role, it is unclear whether binding of a single Ca(V)beta, or multiple Ca(V)betas, to an alpha(1) subunit governs the two distinct functions. Conventional experiments utilizing coexpression of alpha(1) and Ca(V)beta 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)beta(2b) to the carboxyl terminus of alpha(1C) (Ca(V)1.2), creating alpha(1C).beta(2b). Recombinant L-type channels reconstituted in HEK 293 cells with alpha(1C).beta(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).beta(2b) fully restored channel trafficking to the plasma membrane. Co-transfecting Ca(V)beta(2a) with alpha(1C).beta(2b) had little further impact on function. To rule out the possibility that fused Ca(V)beta(2b) was interacting in trans with neighbouring alpha(1) molecules, alpha(1C).beta(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)beta with a pore-forming alpha(1) subunit captures the functional essence of HVA calcium channels, and introduce alpha(1)-Ca(V)beta fusion proteins as a powerful new tool to probe structure-function mechanisms.
Dalton
Stanislava
S
Calcium Signals Laboratory, Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Takahashi
Shoji X
SX
Miriyala
Jayalakshmi
J
Colecraft
Henry M
HM
D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D004553:Electric Conductivity; D006801:Humans; D015640:Ion Channel Gating; D008564:Membrane Potentials; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D017354:Point Mutation; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000502:physiology
C079146:CACNA1C protein, human; C107597:CACNB2 protein, human; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins