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J Biol Chem. 2000 Oct 27;275(43):33704-11.

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

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  • 1Department of Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York 14642, USA.


The current study provides biochemical and functional evidence that the targeting of protein kinase A (PKA) to sites of localized Ca(2+) release confers rapid, specific phosphoregulation of Ca(2+) signaling in pancreatic acinar cells. Regulatory control of Ca(2+) release by PKA-dependent phosphorylation of inositol 1,4, 5-trisphosphate (InsP(3)) receptors was investigated by monitoring Ca(2+) dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP(3) prior to and following PKA activation. Ca(2+) dynamics were imaged with high temporal resolution by digital imaging and electrophysiological methods. The whole cell patch clamp technique was used to introduce caged compounds and to record the activity of a Ca(2+)-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, PKA activators had no significant inhibitory effect on the activation of Cl(-) current evoked by uncaging Ca(2+) or by the photolytic release of higher concentrations of InsP(3). Treatment with Rp-adenosine-3',5'-cyclic monophoshorothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to disrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca(2+) release. Further evidence for the targeting of PKA to the sites of Ca(2+) mobilization was revealed using immunocytochemical methods demonstrating that the R(IIbeta) subunit of PKA was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the type 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 which distinct oscillatory Ca(2+) patterns can be shaped.

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