Proteolytic Cleavage of the Hydrophobic Domain in the CaV{alpha}2{delta}1 Subunit improves assembly and activity of cardiac CaV1.2 Channels [Molecular Biophysics]

Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVβ, and CaVα2δ1 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 PI-PLC, a phospholipase C specific for the cleavage of glycosyl-phosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that C-terminally cleaved CaVα2δ1 proteins upregulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current upregulation.CaVα2δ1 mutants G1060I and G1061I near-eliminated the cell-surface fluorescence of CaVα2δ1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaVα2δ1-mediated increase in peak current density and modulation of the voltage-dependent gating of CaV1.2. These impacts were specific to substitutions with isoleucine residues since functional modulation was partially preserved in CaVα2δ1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaVα2δ1 G1060I and G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 G1060A, failed to coimmunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaVα2δ1 subunit at the predicted membrane interface and further suggest that preventing GPI-anchoring of CaVα2δ1 averts its cell-surface expression, its interaction with CaVα1, and modulation of CaV1.2 currents.

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