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 Ca(2+)-independent fashion [Cardy and Taylor (1998) Biochem. J. 334, 447-455], and Ca(2+) 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(3) receptors, we show that InsP(3)-evoked Ca(2+) mobilization from each is similarly inhibited by calmodulin. In SH-SY5Y cells, which express largely type-1 receptors, calmodulin (IC(50) approximately 15 microM) inhibited InsP(3)-evoked Ca(2+) release only in the presence of Ca(2+). The inhibition was unaffected by calcineurin inhibitors. The effect of calmodulin did not result from enhanced metabolism of InsP(3) because calmodulin also decreased the sensitivity of the Ca(2+) 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 Ca(2+)-calmodulin. Using a scintillation proximity assay to measure (125)I-calmodulin binding to glutathione S-transferase-fusion proteins, we identified two regions of the type-1 InsP(3) receptor (cyt1, residues -6 to 159; and cyt11, residues 1499-1649) that bound (125)I-calmodulin. The higher-affinity site (cyt11) was also photoaffinity labelled with N-hydroxysuccinimidyl-4-azidobenzoate (HSAB)-calmodulin. We speculate that Ca(2+)-independent binding of calmodulin to a site within the first 159 residues of the type-1 InsP(3) receptor inhibits InsP(3) binding and may thereby regulate the kinetics of Ca(2+) release. Ca(2+)-dependent inhibition of Ca(2+) release by calmodulin is mediated by a different site: it may reside on an accessory protein that associates with all three receptor subtypes, or Ca(2+)-calmodulin binding to a site lying between residues 1499 and 1649 of the type-1 receptor may inhibit Ca(2+) release from any tetrameric receptor that includes a type-1 subunit.
Adkins
C E
CE
Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K. cwt1000@cam.ac.uk
Morris
S A
SA
De Smedt
H
H
Sienaert
I
I
Török
K
K
Taylor
C W
CW
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008099:Liver / Q000166:cytology / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000145:classification / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015220:Calcium Channels; D002147:Calmodulin; C506926:ITPR1 protein, human; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; C085029:adenophostin A; D017868:Cyclic AMP-Dependent Protein Kinases; D000241:Adenosine; D002118:Calcium