Targeted phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca2+ release and shapes oscillatory Ca2+ signals

Current study provides biochemical and functional evidence that the targeti ng of protein kinase A (PBA) to sites of localized Ca2+ release confers rap id, specific phosphoregulation of Ca2+ signaling in pancreatic acinar cells , Regulatory control of Ca2+ release by PKA-dependent phosphorylation of in ositol 1,4,5-trisphosphate (InsP(3)) receptors was investigated by monitori ng Ca2+ dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP(3) prior to and following PRA activation, Ca2+ dynamics were imaged with high temporal resolution by digital imaging and electrophysiolo gical methods. The whole cell patch clamp technique was used to introduce c aged compounds and to record the activity of a Ca2+-activated Cl- current, Photolysis of low concentrations of caged InsP(3) evoked Cl- currents that were inhibited by treatment with dibutryl-cAMP or forskolin. In contrast, P KA activators had no significant inhibitory effect on the activation of Cl- current evoked by uncaging Ca2+ or by the photolytic release of higher con centrations of InsP(3). Treatment with Rp-adenosine-3',5'-cyclic monophosho rothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to d isrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca2+ release. Further evidence for the targeting of PKA to the sites of Ca2+ mobilization was revealed using immunocytochemical methods demonstrat ing that the R-II beta subunit of PKA. was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the typ e I or type II InsP(3) receptors, Finally, we demonstrate that the pattern of signaling evoked by acetylcholine can be converted to one that is more " CCK-like" by raising cAMP levels. Our data provide a simple mechanism by wh ich distinct oscillatory Ca2+ patterns can he shaped.