Proteolytic cleavage of the hydrophobic domain in the CaVα2δ1 subunit improves assembly and activity of cardiac CaV1.2 channels

Voltage-gated L-type Ca(V)1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming Ca-V alpha 1, Ca-V beta, and Ca-V alpha 2 delta 1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca2+ currents, but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac Ca-V alpha 2 delta 1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved Ca-V alpha 2 delta 1 proteins up-regulate Ca(V)1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (Ca-V alpha 2 delta 1 Delta 1059-1063) impaired current up-regulation. Ca-V alpha 2 delta 1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of Ca-V alpha 2 delta 1, indicated by two-color flow cytometry assays and confocal imaging, and prevented Ca-V alpha 2 delta 1-mediated increase in peak current density and modulation of the voltage-dependent gating of Ca(V)1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in Ca-V alpha 2 delta 1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of Ca-V alpha 2 delta 1 G1060I and Ca-V alpha 2 delta 1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, Ca-V alpha 2 delta 1 Delta 1059-1063, but not Ca-V alpha 2 delta 1 G1060A, failed to co-immunoprecipitate with Ca(V)1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the Ca-V alpha 2 delta 1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of Ca-V alpha 2 delta 1 averts its cell-surface expression, its interaction with Ca-V alpha 1, and modulation of Ca(V)1.2 currents.