Dataset Information

Proteolytic processing of the L-type Ca ²⁺ channel alpha ₁1.2 subunit in neurons.

ABSTRACT: Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α ₁1.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α ₁1.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Methods: To better determine the apparent molecular masses (M _R)of the α ₁1.2 size forms, extensive systematic immunoblotting of brain tissue as well as full length and C-terminally truncated α ₁1.2 expressed in HEK293 cells was conducted using six different region-specific antibodies against α ₁1.2. Results: The full length form of α ₁1.2 migrated, as expected, with an apparent M _R of ~250 kDa. A shorter form of comparable prevalence with an apparent M _R of ~210 kDa could only be detected in immunoblots probed with antibodies recognizing α ₁1.2 at an epitope 400 or more residues upstream of the C-terminus. Conclusions: The main two size forms of α ₁1.2 are the full length form and a shorter form, which lacks ~350 distal C-terminal residues. Midchannel cleavage as suggested by Michailidis et al. (2014) is at best minimal in brain tissue.

SUBMITTER: Buonarati OR

PROVIDER: S-EPMC5531164 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

Proteolytic processing of the L-type Ca 2+ channel alpha 11.2 subunit in neurons.

Buonarati Olivia R OR Henderson Peter B PB Murphy Geoffrey G GG Horne Mary C MC Hell Johannes W JW

F1000Research 20170721

Background: The L-type Ca2+ channel Cav1.2 is a prominent regulator of neuronal excitability, synaptic plasticity, and gene expression. The central element of Cav1.2 is the pore-forming α 11.2 subunit. It exists in two major size forms, whose molecular masses have proven difficult to precisely determine. Recent work suggests that α 11.2 is proteolytically cleaved between the second and third of its four pore-forming domains (Michailidis et al,. 2014). Metho ...[more]

PMID: 28781760

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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

Proteolytic processing of the L-type Ca ²⁺ channel alpha ₁1.2 subunit in neurons.

Publications

Proteolytic processing of the L-type Ca <sup>2+</sup> channel alpha <sub>1</sub>1.2 subunit in neurons.

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