CAMP-DEPENDENT PHOSPHORYLATION OF THE CARDIAC-TYPE ALPHA-1 SUBUNIT OFTHE VOLTAGE-DEPENDENT CA2-CELL LINE( CHANNEL IN A MURINE PANCREATIC BETA)

Two voltage-dependent calcium channels (VDCCs) have been reported in p ancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 c ells, a murine pancreatic beta-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isob utyl-3-methylxanthine (IBMX) and forskolin, agonists that elevate cAMP in these cells, on the phosphorylation of this subunit in intact beta TC3 cells using a sensitive back-phosphorylation technique. This tech nique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 c ells with forskolin or IBMX resulted in the phosphorylation of the car diac-type alpha 1 subunit as evidenced by a 40-60% decrease in the abi lity of the 257-kDa form to serve as a substrate in the in vitro back- phosphorylation reaction with [gamma-P-32]ATP and the catalytic subuni t of cAMP-dependent protein kinase (PKA). The effects of forskolin wer e time- and concentration-dependent. The concentration-dependency of f orskolin-induced phosphorylation of the cardiac-type alpha 1 subunit a nd the potentiation of glucose-induced insulin secretion were highly c orrelated, a finding that is consistent with a role for such phosphory lation in mediating at least some of the effects of cAMP on secretion.