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

InsP₃ binding to type-1, but not type-3, InsP₃ 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 InsP₃ 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 InsP₃ receptors, we show that InsP₃-evoked Ca²⁺ mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC₅₀ ≈ 15 μM) inhibited InsP₃-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 InsP₃ because calmodulin also decreased the sensitivity of the Ca²⁺ stores to adenophostin A, a non-metabolizable InsP₃-receptor agonist. Protein kinase A-catalysed phosphorylation of type-1 InsP₃ receptors was unaffected by Ca²⁺-calmodulin. Using a scintillation proximity assay to measure ¹²⁵I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP₃ receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound ¹²⁵I-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 InsP₃ receptor inhibits InsP₃ 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.