Differential phosphorylation of two size forms of the neuronal class C L-type calcium channel alpha 1 subunit.
L-type calcium channels mediate long-lasting calcium currents which are modulated by protein phosphorylation. Using site-directed anti-peptide antibodies, we show that the alpha 1 subunit of the neuronal class C L-type calcium channel from rat brain exists in two size forms. The longer form, LC2, with an apparent molecular mass of 210-235 kDa was phosphorylated in vitro by cAMP-dependent protein kinase (cA-PK), but the shorter form, LC1, with an apparent molecular mass of 190-195 kDa was not a substrate for cA-PK. In contrast, LC1 and LC2 are both substrates for protein kinase C (PKC), calcium- and calmodulin-dependent protein kinase II, and cGMP-dependent protein kinase (cG-PK). The site-directed anti-peptide antibody CNC2 was produced against the COOH-terminal end of the class C L-type alpha 1 subunit as predicted by molecular cloning and sequencing of cDNA. CNC2 recognized LC2 but not LC1 by immunoblotting and immunoprecipitated only LC2 phosphorylated by either cA-PK or PKC. These results indicate that LC1 is truncated at its COOH-terminal end with respect to LC2 and that cA-PK preferentially phosphorylates sites in the COOH-terminal region of the alpha 1 subunit that are present in LC2 but not LC1. The selectivity of cA-PK for phosphorylation of the COOH-terminal region of LC2 suggests that the channel activities of the two alpha 1 subunit size forms may be differentially regulated by neurotransmitters and hormones which act through cAMP-dependent mechanisms, while both alpha 1 subunit isoforms may be modulated by PKC, cG-PK, and calcium- and calmodulin-dependent protein kinase II.
Hell
J W
JW
Department of Pharmacology, University of Washington, Seattle 98195.
Yokoyama
C T
CT
Wong
S T
ST
Warner
C
C
Snutch
T P
TP
Catterall
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D001921:Brain / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA; D015151:Immunoblotting; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D009474:Neurons / Q000378:metabolism*; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D000906:Antibodies; D000936:Antigen-Antibody Complex; D015220:Calcium Channels; D046911:Macromolecular Substances; D008565:Membrane Proteins; D010455:Peptides; D004247:DNA; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases