Proteolytic cleavage of the hydrophobic domain in the CaV?2?1 subunit improves assembly and activity of cardiac CaV1.2 channels.

ABSTRACT: Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaV?1, CaV?, and CaV?2?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 CaV?2?1 prompting us to investigate deletions of its hydrophobic transmembrane domain. Patch-clamp experiments indicated that the C-terminally cleaved CaV?2?1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaV?2?1 ?1059-1063) impaired current up-regulation. CaV?2?1 mutants G1060I and G1061I nearly eliminated the cell-surface fluorescence of CaV?2?1, indicated by two-color flow cytometry assays and confocal imaging, and prevented CaV?2?1-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 because functional modulation was partially preserved in CaV?2?1 G1060A and G1061A proteins. Moreover, C-terminal fragments exhibited significantly altered mobility in denatured immunoblots of CaV?2?1 G1060I and CaV?2?1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaV?2?1 ?1059-1063, but not CaV?2?1 G1060A, failed to co-immunoprecipitate with CaV1.2. Altogether, our data support a model in which small neutral hydrophobic residues facilitate the post-translational cleavage of the CaV?2?1 subunit at the predicted membrane interface and further suggest that preventing GPI anchoring of CaV?2?1 averts its cell-surface expression, its interaction with CaV?1, and modulation of CaV1.2 currents.

SUBMITTER: Segura E 

PROVIDER: S-EPMC5491792 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature