CAMP-dependent phosphorylation of the cardiac-type alpha 1 subunit of the voltage-dependent Ca2+ channel in a murine pancreatic beta-cell line.

Abstract

Two voltage-dependent calcium channels (VDCCs) have been reported in pancreatic islets: the beta-cell/endocrine-brain and cardiac subtypes. The cardiac-type alpha 1 subunit was isolated from cultured beta TC3 cells, a murine pancreatic beta-cell line, by immunoprecipitation with a specific polyclonal antibody. We have examined the effects of 1-isobutyl-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 technique allows quantitative detection of protein phosphorylation that is specifically stimulated by cAMP. The stimulation of intact beta TC3 cells with forskolin or IBMX resulted in the phosphorylation of the cardiac-type alpha 1 subunit as evidenced by a 40-60% decrease in the ability of the 257-kDa form to serve as a substrate in the in vitro back-phosphorylation reaction with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase (PKA). The effects of forskolin were time- and concentration-dependent. The concentration-dependency of forskolin-induced phosphorylation of the cardiac-type alpha 1 subunit and the potentiation of glucose-induced insulin secretion were highly correlated, a finding that is consistent with a role for such phosphorylation in mediating at least some of the effects of cAMP on secretion.