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

Abstract

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.