IMMUNOCHEMICAL IDENTIFICATION AND DIFFERENTIAL PHOSPHORYLATION OF ALTERNATIVELY SPLICED FORMS OF THE ALPHA(1A) SUBUNIT OF BRAIN CALCIUM CHANNELS

Biochemical properties of the alpha(1) subunits of class A brain calci um channels (alpha(1A)) were examined in adult rat brain membrane frac tions using a site-directed antipeptide antibody (anti-CNA3) specific for alpha(1A). Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (K-d similar to 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-co notoxin GVIA. In immunoblotting and immunoprecipitation experiments, a nti-CNA3 recognized at least two distinct immunoreactive alpha(1A) pol ypeptides, 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 alpha(1A) polypeptides were also specifically re cognized by both anti-BI-Nt and anti-HI-1-Ct antibodies, which are dir ected against the NH2- and COOH-terminal ends of alpha(1A) predicted f rom cDNA sequence, respectively. These data indicate that the predicte d NH2 and COOH termini are present in both size forms and therefore th at these isoforms of alpha(1A) are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH2 or C OOH termini. The 220-kDa form was phosphorylated preferentially by cAM P-dependent protein kinase, whereas protein kinase C and cGMP-dependen t protein kinase preferentially phosphorylated the 190-kDa form. Our r esults identify at least two distinct alpha(1A) subunits with differen t molecular mass, demonstrate that they may result from alternative mR NA splicing, and suggest that they may be differentially regulated by protein phosphorylation.