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 calcium channels (alpha 1A) were examined in adult rat brain membrane fractions using a site-directed anti-peptide antibody (anti-CNA3) specific for alpha 1A. Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for omega-conotoxin MVIIC (Kd approximately 100 pM), but not receptor sites for the dihydropyridine isradipine or for omega-conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive alpha 1A 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 alpha 1A 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 alpha 1A 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 alpha 1A 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 alpha 1A 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.
Sakurai
T
T
Department of Pharmacology, University of Washington, Seattle 98195, USA.
Hell
J W
JW
Woppmann
A
A
Miljanich
G P
GP
Catterall
W A
WA
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D004095:Dihydropyridines / Q000378:metabolism; D004789:Enzyme Activation; D007120:Immunochemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems; D020866:omega-Conotoxin GVIA; D020960:omega-Conotoxins
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D010455:Peptides; D020960:omega-Conotoxins; C075851:omega-conotoxin-MVIIC; D020866:omega-Conotoxin GVIA; D011494:Protein Kinases