Immunochemical identification and differential phosphorylation of alternatively spliced forms of the α1A subunit of brain calcium channels

Takashi Sakurai, Johannes W Hell, Andreas Woppmann, George P. Miljanich, William A. Catterall

Research output: Contribution to journalArticle

39 Scopus citations

Abstract

Biochemical properties of the α1 subunits of class A brain calcium channels (α1A) were examined in adult rat brain membrane fractions using a site-directed antipeptide antibody (anti-CNA3) specific for α1A. AntiCNA3 specifically immunoprecipitated high affinity receptor sites for ω-conotoxin MVIIC (Kd ∼ 100 PM), but not receptor sites for the dihydropyridine isradipine or for ω-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive α1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220- and 190-kDa α1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH2- and COOH-terminal ends of α1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH2 and COOH termini are present in both size forms and therefore that these isoforms of α1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct α1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.

Original languageEnglish (US)
Pages (from-to)21234-21242
Number of pages9
JournalJournal of Biological Chemistry
Volume270
Issue number36
StatePublished - Sep 8 1995
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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