Proteolytic cleavage of the hydrophobic domain in the Caα2δ1 subunit improves assembly and activity of cardiac Ca1.2 channels.

Voltage-gated L-type Ca1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming Caα1, Caβ, and Caα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca 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 Caα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved Caα2δ1 proteins up-regulate Ca1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (Caα2δ1 Δ1059-1063) impaired current up-regulation. Caα2δ1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of Caα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented Caα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of Ca1.2. These impacts were specific to substitutions with isoleucine residues because functional modulation was partially preserved in Caα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of Caα2δ1 G1060I and Caα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, Caα2δ1 Δ1059-1063, but not Caα2δ1 G1060A, failed to co-immunoprecipitate with Ca1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the Caα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of Caα2δ1 averts its cell-surface expression, its interaction with Caα1, and modulation of Ca1.2 currents.