Ca2+-calmodulin inhibits Ca2+ release mediated by type-1,-2 and-3 inositol trisphosphate receptors

InsP(3) binding to type-1,but not type-3, InsP(3) receptors is inhibited by calmodulin in a Ca2+-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca2+ mobilization by type-1 InsP(3) receptors of cerebellum is inhibited by calmodulin [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 11627-11632]. Using cell types expressing predominantly type-1, -2 or -3 InsP, receptors, we show that InsP,evoked Ca2+ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC50 approximate to 15 mu M) inhibited InsP(3)-evoked Ca2+ release only in the presence of Ca2+. The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP, because calmodulin also decreased the sensitivity of the Ca2+ stores to adenophostin A, a non-metabolizable InsP(3)-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP(3) receptors was unaffected by Ca2+-calmodulin. Using a scintillation proximity assay to measure I-125-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP, receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound I-125-calmodulin. The higher-affinity site(cyt11)was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca2+-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP(3) receptor inhibits InsP, binding and may thereby regulate the kinetics of Ca2+ release. Ca2+-dependent inhibition of Ca2+ release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca2+ calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca2+ release from any tetrameric receptor that includes a type-1 subunit.