Specific phosphorylation of a site in the full-length form of the alpha 1 subunit of the cardiac L-type calcium channel by adenosine 3',5'-cyclic monophosphate-dependent protein kinase.
Voltage-gated L-type Ca2+ channels mediate Ca2+ entry into cells in response to membrane depolarization. Ca2+ entry through the cardiac Ca2+ channel determines the rate and force of contraction, and modulation of Ca2+ channel activity by beta-adrenergic agents acting through adenosine 3',5'-cyclic monophosphate-(cAMP)-dependent protein phosphorylation contributes to physiological regulation of cardiac function by the sympathetic nervous system. Immunoblotting experiments using site-directed anti-peptide antibodies against different peptide segments indicate that the alpha 1 subunit of the cardiac L-type Ca2+ channel exists in two size forms with apparent molecular masses of 240 and 210 kDa, which we call alpha 1(242) and alpha 1(210), Alpha 1(242) corresponds to the full-length cardiac alpha 1 subunit predicted from its cDNA sequence, while alpha 1(210) is truncated at its COOH terminus. Only alpha 1(242) is phosphorylated in vitro by cAMP-dependent protein kinase. Protein microsequencing and peptide mapping of wild-type and mutant fusion proteins show that this phosphorylation occurs at serine 1928 near the COOH terminus. Phosphorylation of this residue can be detected by phosphospecific antibodies raised against the corresponding phosphopeptide. Experiments with these antibodies show that alpha 1(242) is phosphorylated in intact cells expressing the cardiac alpha 1 subunit in response to increased intracellular levels of cAMP. These results identify serine 1928 on the alpha 1 subunit as a possible site of regulation by cAMP-dependent phosphorylation.
De Jongh
K S
KS
Department of Pharmacology, University of Washington, Seattle 98195-7280, USA.
Murphy
B J
BJ
Colvin
A A
AA
Hell
J W
JW
Takahashi
M
M
Catterall
W A
WA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009206:Myocardium / Q000378:metabolism*; D010449:Peptide Mapping; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D015220:Calcium Channels; D010455:Peptides; D011994:Recombinant Proteins; D017868:Cyclic AMP-Dependent Protein Kinases