The various inositol 1,4,5-trisphosphate receptor (IP3R) isoforms are potential substrates for several protein kinases. We compared the in vitro phosphorylation of purified IP(3)R1 and IP(3)R3 by the catalytic subunit of protein kinase C (PKC). Phosphorylation of IP(3)R1 by PKC was about eight times stronger than that Of IP3R3 under identical conditions. Protein kinase A strongly stimulated the PKC-induced phosphorylation of IP(3)R1. In contrast, Ca2+ inhibited its phosphorylation (IC50 less than or equal to 2 muM) and this inhibition was further potentiated by calmodulin (CaM), while the Ca2+-independent CaM mutant CaM1234 was ineffective. Ca2+ and CaM, however, did not inhibit IP(3)R3 phosphorylation by PKC. Taken together, these findings show that Ca2+ and CaM differentially regulate the PKC-mediated phosphorylation of IP(3)R1 and IP(3)R3 and are indicative for a role for the inhibition of IP(3)R1 phosphorylation by Ca2+ and CaM in the negative slope of the bell-shaped effect of Ca2+ on IP3R function. (C) 2004 Elsevier Inc. All rights reserved.