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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

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

Segura Emilie E Bourdin Benoîte B Tétreault Marie-Philippe MP Briot Julie J Allen Bruce G BG Mayer Gaétan G Parent Lucie L

The Journal of biological chemistry 20170511 26

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 pho ...[more]

PMID: 28495885

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Project description:Mammalian circadian (24 h) rhythms are timed by the pattern of spontaneous action potential firing in the suprachiasmatic nucleus (SCN). This oscillation in firing is produced through circadian regulation of several membrane currents, including large-conductance Ca2+- and voltage-activated K+ (BK) and L-type Ca2+ channel (LTCC) currents. During the day steady-state BK currents depend mostly on LTCCs for activation, whereas at night they depend predominantly on ryanodine receptors (RyRs). However, the contribution of these Ca2+ channels to BK channel activation during action potential firing has not been thoroughly investigated. In this study, we used a pharmacological approach to determine that both LTCCs and RyRs contribute to the baseline membrane potential of SCN action potential waveforms, as well as action potential-evoked BK current, during the day and night, respectively. Since the baseline membrane potential is a major determinant of circadian firing rate, we focused on the LTCCs contributing to low voltage activation of BK channels during the subthreshold phase. For these experiments, two LTCC subtypes found in SCN (CaV1.2 and CaV1.3) were coexpressed with BK channels in heterologous cells, where their differential contributions could be separately measured. CaV1.3 channels produced currents that were shifted to more hyperpolarized potentials compared with CaV1.2, resulting in increased subthreshold Ca2+ and BK currents during an action potential command. These results show that although multiple Ca2+ sources in SCN can contribute to the activation of BK current during an action potential, specific BK-CaV1.3 partnerships may optimize the subthreshold BK current activation that is critical for firing rate regulation.NEW & NOTEWORTHY BK K+ channels are important regulators of firing. Although Ca2+ channels are required for their activation in excitable cells, it is not well understood how BK channels activate using these Ca2+ sources during an action potential. This study demonstrates the differences in BK current activated by CaV1.2 and CaV1.3 channels in clock neurons and heterologous cells. The results define how specific ion channel partnerships can be engaged during distinct phases of the action potential.

Dataset Information

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

Publications

Proteolytic cleavage of the hydrophobic domain in the Ca<sub>V</sub>α2δ1 subunit improves assembly and activity of cardiac Ca<sub>V</sub>1.2 channels.

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