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A three-way inter-molecular network accounts for the CaV?2?1-induced functional modulation of the pore-forming CaV1.2 subunit.

ABSTRACT: L-type CaV1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming CaV?1C assembled with CaV? and CaV?2?1 subunits. A direct interaction between CaV?2?1 and Asp-181 in the first extracellular loop of CaV?1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of CaV?2?1 complexed with the voltage sensor domain of CaV?1C suggests that Ser-261 and Ser-263 residues in the metal ion-dependent adhesion site (MIDAS) motif are determinant in this interaction, but this hypothesis is untested. Here, coimmunoprecipitation assays and patch-clamp experiments of single-substitution variants revealed that CaV?2?1 Asp-259 and Ser-261 are the two most important residues in regard to protein interactions and modulation of CaV1.2 currents. In contrast, mutating the side chains of CaV?2?1 Ser-263, Thr-331, and Asp-363 with alanine did not completely prevent channel function. Molecular dynamics simulations indicated that the carboxylate side chain of CaV?2?1 Asp-259 coordinates the divalent cation that is further stabilized by the oxygen atoms from the hydroxyl side chain of CaV?2?1 Ser-261 and the carboxylate group of CaV?1C Asp-181. In return, the hydrogen atoms contributed by the side chain of Ser-261 and the main chain of Ser-263 bonded the oxygen atoms of CaV1.2 Asp-181. We propose that CaV?2?1 Asp-259 promotes Ca2+ binding necessary to produce the conformation of the VWA domain that locks CaV?2?1 Ser-261 and Ser-263 within atomic distance of CaV?1C Asp-181. This three-way network appears to account for the CaV?2?1-induced modulation of CaV1.2 currents.

SUBMITTER: Briot J

PROVIDER: S-EPMC5950009 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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A three-way inter-molecular network accounts for the CaVα2δ1-induced functional modulation of the pore-forming CaV1.2 subunit.

Briot Julie J Mailhot Olivier O Bourdin Benoîte B Tétreault Marie-Philippe MP Najmanovich Rafael R Parent Lucie L

The Journal of biological chemistry 20180327 19

L-type CaV1.2 channels are essential for the excitation-contraction coupling in cardiomyocytes and are hetero-oligomers of a pore-forming CaVα1C assembled with CaVβ and CaVα2δ1 subunits. A direct interaction between CaVα2δ1 and Asp-181 in the first extracellular loop of CaVα1 reproduces the native properties of the channel. A 3D model of the von Willebrand factor type A (VWA) domain of CaVα2δ1 complexed with the voltage ...[more]

PMID: 29588365

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Project description: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 phosphatidylinositol-specific phospholipase C, a phospholipase C specific for the cleavage of glycosylphosphatidylinositol (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 the C-terminally cleaved CaVα2δ1 proteins up-regulate CaV1.2 channels. In contrast, deleting the residues before the single hydrophobic segment (CaVα2δ1 Δ1059-1063) impaired current up-regulation. CaVα2δ1 mutants G1060I and G1061I nearly 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 because 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 CaVα2δ1 G1061I, suggesting that these mutant proteins were impaired in proteolytic processing. Finally, CaVα2δ1 Δ1059-1063, but not CaVα2δ1 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 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.

Dataset Information

A three-way inter-molecular network accounts for the CaV?2?1-induced functional modulation of the pore-forming CaV1.2 subunit.

Publications

A three-way inter-molecular network accounts for the Ca<sub>V</sub>α2δ1-induced functional modulation of the pore-forming Ca<sub>V</sub>1.2 subunit.

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