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. 1998 Sep 1;334(Pt 2):447–455. doi: 10.1042/bj3340447

A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors.

T J Cardy 1, C W Taylor 1
PMCID: PMC1219708  PMID: 9716504

Abstract

Calmodulin inhibits both inositol 1,4,5-trisphosphate (IP3) binding to, and IP3-evoked Ca2+ release by, cerebellar IP3 receptors [Patel, Morris, Adkins, O'Beirne and Taylor (1997) Proc. Natl. Acad. Sci. U. S.A. 94, 11627-11632]. In the present study, full-length rat type-1 and -3 IP3 receptors were expressed at high levels in insect Spodoptera frugiperda 9 cells and the effects of calmodulin were examined. In the absence of Ca2+, calmodulin caused a concentration-dependent and reversible inhibition of [3H]IP3 binding to type-1 IP3 receptors by decreasing their apparent affinity for IP3. The effect was not reproduced by high concentrations of troponin C, parvalbumin or S-100. Increasing the medium free [Ca2+] ([Ca2+]m) inhibited [3H]IP3 binding to type-1 receptors, but the further inhibition caused by a submaximal concentration of calmodulin was similar at each [Ca2+]m. In the absence of Ca2+, 125I-calmodulin bound to a single site on each type-1 receptor subunit and to an additional site in the presence of Ca2+. There was no detectable binding of 125I-calmodulin to type-3 receptors and binding of [3H]IP3 was insensitive to calmodulin at all [Ca2+]m. Both peptide and conventional Ca2+-calmodulin antagonists affected neither [3H]IP3 binding directly nor the inhibitory effect of calmodulin in the absence of Ca2+, but each caused a [Ca2+]m-dependent reversal of the inhibition of [3H]IP3 binding caused by calmodulin. Camstatin, a peptide that binds to calmodulin equally well in the presence or absence of Ca2+, reversed the inhibitory effects of calmodulin on [3H]IP3 binding at all [Ca2+]m. We conclude that calmodulin specifically inhibits [3H]IP3 binding to type-1 IP3 receptors: the first example of a protein regulated by calmodulin in an entirely Ca2+-independent manner. Inhibition of type-1 IP3 receptors by calmodulin may dynamically regulate their sensitivity to IP3 in response to the changes in cytosolic free calmodulin concentration thought to accompany stimulation of neurones.

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Selected References

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  1. Arnon A., Cook B., Gillo B., Montell C., Selinger Z., Minke B. Calmodulin regulation of light adaptation and store-operated dark current in Drosophila photoreceptors. Proc Natl Acad Sci U S A. 1997 May 27;94(11):5894–5899. doi: 10.1073/pnas.94.11.5894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bennett D. L., Petersen C. C., Cheek T. R. Calcium signalling. Cracking ICRAC in the eye. Curr Biol. 1995 Nov 1;5(11):1225–1228. doi: 10.1016/s0960-9822(95)00243-0. [DOI] [PubMed] [Google Scholar]
  3. Blondel O., Takeda J., Janssen H., Seino S., Bell G. I. Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues. J Biol Chem. 1993 May 25;268(15):11356–11363. [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Callamaras N., Parker I. Inositol 1,4,5-trisphosphate receptors in Xenopus laevis oocytes: localization and modulation by Ca2+. Cell Calcium. 1994 Jan;15(1):66–78. doi: 10.1016/0143-4160(94)90105-8. [DOI] [PubMed] [Google Scholar]
  6. Cameron A. M., Steiner J. P., Roskams A. J., Ali S. M., Ronnett G. V., Snyder S. H. Calcineurin associated with the inositol 1,4,5-trisphosphate receptor-FKBP12 complex modulates Ca2+ flux. Cell. 1995 Nov 3;83(3):463–472. doi: 10.1016/0092-8674(95)90124-8. [DOI] [PubMed] [Google Scholar]
  7. Carafoli E. The Ca2+ pump of the plasma membrane. J Biol Chem. 1992 Feb 5;267(4):2115–2118. [PubMed] [Google Scholar]
  8. Cardy T. J., Traynor D., Taylor C. W. Differential regulation of types-1 and -3 inositol trisphosphate receptors by cytosolic Ca2+. Biochem J. 1997 Dec 15;328(Pt 3):785–793. doi: 10.1042/bj3280785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chen S. R., MacLennan D. H. Identification of calmodulin-, Ca(2+)-, and ruthenium red-binding domains in the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1994 Sep 9;269(36):22698–22704. [PubMed] [Google Scholar]
  10. Cheney R. E., Mooseker M. S. Unconventional myosins. Curr Opin Cell Biol. 1992 Feb;4(1):27–35. doi: 10.1016/0955-0674(92)90055-h. [DOI] [PubMed] [Google Scholar]
  11. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  12. Cheung W. Y. Calmodulin plays a pivotal role in cellular regulation. Science. 1980 Jan 4;207(4426):19–27. doi: 10.1126/science.6243188. [DOI] [PubMed] [Google Scholar]
  13. Cox J. A. Interactive properties of calmodulin. Biochem J. 1988 Feb 1;249(3):621–629. doi: 10.1042/bj2490621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Danoff S. K., Supattapone S., Snyder S. H. Characterization of a membrane protein from brain mediating the inhibition of inositol 1,4,5-trisphosphate receptor binding by calcium. Biochem J. 1988 Sep 15;254(3):701–705. doi: 10.1042/bj2540701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ehlers M. D., Zhang S., Bernhadt J. P., Huganir R. L. Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell. 1996 Mar 8;84(5):745–755. doi: 10.1016/s0092-8674(00)81052-1. [DOI] [PubMed] [Google Scholar]
  16. Geiser J. R., van Tuinen D., Brockerhoff S. E., Neff M. M., Davis T. N. Can calmodulin function without binding calcium? Cell. 1991 Jun 14;65(6):949–959. doi: 10.1016/0092-8674(91)90547-c. [DOI] [PubMed] [Google Scholar]
  17. Gnegy M. E. Calmodulin in neurotransmitter and hormone action. Annu Rev Pharmacol Toxicol. 1993;33:45–70. doi: 10.1146/annurev.pa.33.040193.000401. [DOI] [PubMed] [Google Scholar]
  18. Graff J. M., Young T. N., Johnson J. D., Blackshear P. J. Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C. J Biol Chem. 1989 Dec 25;264(36):21818–21823. [PubMed] [Google Scholar]
  19. Greenlee D. V., Andreasen T. J., Storm D. R. Calcium-independent stimulation of Bordetella pertussis adenylate cyclase by calmodulin. Biochemistry. 1982 May 25;21(11):2759–2764. doi: 10.1021/bi00540a028. [DOI] [PubMed] [Google Scholar]
  20. Hanson P. I., Schulman H. Neuronal Ca2+/calmodulin-dependent protein kinases. Annu Rev Biochem. 1992;61:559–601. doi: 10.1146/annurev.bi.61.070192.003015. [DOI] [PubMed] [Google Scholar]
  21. Houdusse A., Silver M., Cohen C. A model of Ca(2+)-free calmodulin binding to unconventional myosins reveals how calmodulin acts as a regulatory switch. Structure. 1996 Dec 15;4(12):1475–1490. doi: 10.1016/s0969-2126(96)00154-2. [DOI] [PubMed] [Google Scholar]
  22. Joseph S. K. The inositol triphosphate receptor family. Cell Signal. 1996 Jan;8(1):1–7. doi: 10.1016/0898-6568(95)02012-8. [DOI] [PubMed] [Google Scholar]
  23. Kakiuchi S., Yasuda S., Yamazaki R., Teshima Y., Kanda K., Kakiuchi R., Sobue K. Quantitative determinations of calmodulin in the supernatant and particulate fractions of mammalian tissues. J Biochem. 1982 Oct;92(4):1041–1048. doi: 10.1093/oxfordjournals.jbchem.a134019. [DOI] [PubMed] [Google Scholar]
  24. Lee H. C., Aarhus R., Graeff R. M. Sensitization of calcium-induced calcium release by cyclic ADP-ribose and calmodulin. J Biol Chem. 1995 Apr 21;270(16):9060–9066. doi: 10.1074/jbc.270.16.9060. [DOI] [PubMed] [Google Scholar]
  25. Levin R. M., Weiss B. Binding of trifluoperazine to the calcium-dependent activator of cyclic nucleotide phosphodiesterase. Mol Pharmacol. 1977 Jul;13(4):690–697. [PubMed] [Google Scholar]
  26. Liu M., Chen T. Y., Ahamed B., Li J., Yau K. W. Calcium-calmodulin modulation of the olfactory cyclic nucleotide-gated cation channel. Science. 1994 Nov 25;266(5189):1348–1354. doi: 10.1126/science.266.5189.1348. [DOI] [PubMed] [Google Scholar]
  27. Liu Y. C., Storm D. R. Regulation of free calmodulin levels by neuromodulin: neuron growth and regeneration. Trends Pharmacol Sci. 1990 Mar;11(3):107–111. doi: 10.1016/0165-6147(90)90195-e. [DOI] [PubMed] [Google Scholar]
  28. Maeda N., Kawasaki T., Nakade S., Yokota N., Taguchi T., Kasai M., Mikoshiba K. Structural and functional characterization of inositol 1,4,5-trisphosphate receptor channel from mouse cerebellum. J Biol Chem. 1991 Jan 15;266(2):1109–1116. [PubMed] [Google Scholar]
  29. Mignery G. A., Newton C. L., Archer B. T., 3rd, Südhof T. C. Structure and expression of the rat inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1990 Jul 25;265(21):12679–12685. [PubMed] [Google Scholar]
  30. Mikoshiba K. The InsP3 receptor and intracellular Ca2+ signaling. Curr Opin Neurobiol. 1997 Jun;7(3):339–345. doi: 10.1016/s0959-4388(97)80061-x. [DOI] [PubMed] [Google Scholar]
  31. Nakagawa T., Okano H., Furuichi T., Aruga J., Mikoshiba K. The subtypes of the mouse inositol 1,4,5-trisphosphate receptor are expressed in a tissue-specific and developmentally specific manner. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6244–6248. doi: 10.1073/pnas.88.14.6244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Newton C. L., Mignery G. A., Südhof T. C. Co-expression in vertebrate tissues and cell lines of multiple inositol 1,4,5-trisphosphate (InsP3) receptors with distinct affinities for InsP3. J Biol Chem. 1994 Nov 18;269(46):28613–28619. [PubMed] [Google Scholar]
  33. O'Neil K. T., DeGrado W. F. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. 1990 Feb;15(2):59–64. doi: 10.1016/0968-0004(90)90177-d. [DOI] [PubMed] [Google Scholar]
  34. Patel S., Morris S. A., Adkins C. E., O'Beirne G., Taylor C. W. Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11627–11632. doi: 10.1073/pnas.94.21.11627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Phillips A. M., Bull A., Kelly L. E. Identification of a Drosophila gene encoding a calmodulin-binding protein with homology to the trp phototransduction gene. Neuron. 1992 Apr;8(4):631–642. doi: 10.1016/0896-6273(92)90085-r. [DOI] [PubMed] [Google Scholar]
  36. Porter J. A., Minke B., Montell C. Calmodulin binding to Drosophila NinaC required for termination of phototransduction. EMBO J. 1995 Sep 15;14(18):4450–4459. doi: 10.1002/j.1460-2075.1995.tb00124.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Scott K., Sun Y., Beckingham K., Zuker C. S. Calmodulin regulation of Drosophila light-activated channels and receptor function mediates termination of the light response in vivo. Cell. 1997 Oct 31;91(3):375–383. doi: 10.1016/s0092-8674(00)80421-3. [DOI] [PubMed] [Google Scholar]
  38. Slemmon J. R., Morgan J. I., Fullerton S. M., Danho W., Hilbush B. S., Wengenack T. M. Camstatins are peptide antagonists of calmodulin based upon a conserved structural motif in PEP-19, neurogranin, and neuromodulin. J Biol Chem. 1996 Jul 5;271(27):15911–15917. doi: 10.1074/jbc.271.27.15911. [DOI] [PubMed] [Google Scholar]
  39. Smith D. P., Ranganathan R., Hardy R. W., Marx J., Tsuchida T., Zuker C. S. Photoreceptor deactivation and retinal degeneration mediated by a photoreceptor-specific protein kinase C. Science. 1991 Dec 6;254(5037):1478–1484. doi: 10.1126/science.1962207. [DOI] [PubMed] [Google Scholar]
  40. Sugawara H., Kurosaki M., Takata M., Kurosaki T. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J. 1997 Jun 2;16(11):3078–3088. doi: 10.1093/emboj/16.11.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Supattapone S., Worley P. F., Baraban J. M., Snyder S. H. Solubilization, purification, and characterization of an inositol trisphosphate receptor. J Biol Chem. 1988 Jan 25;263(3):1530–1534. [PubMed] [Google Scholar]
  42. Tripathy A., Xu L., Mann G., Meissner G. Calmodulin activation and inhibition of skeletal muscle Ca2+ release channel (ryanodine receptor). Biophys J. 1995 Jul;69(1):106–119. doi: 10.1016/S0006-3495(95)79880-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Warr C. G., Kelly L. E. Identification and characterization of two distinct calmodulin-binding sites in the Trpl ion-channel protein of Drosophila melanogaster. Biochem J. 1996 Mar 1;314(Pt 2):497–503. doi: 10.1042/bj3140497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wojcikiewicz R. J. Type I, II, and III inositol 1,4,5-trisphosphate receptors are unequally susceptible to down-regulation and are expressed in markedly different proportions in different cell types. J Biol Chem. 1995 May 12;270(19):11678–11683. doi: 10.1074/jbc.270.19.11678. [DOI] [PubMed] [Google Scholar]
  45. Wolenski J. S. Regulation of calmodulin-binding myosins. Trends Cell Biol. 1995 Aug;5(8):310–316. doi: 10.1016/s0962-8924(00)89053-4. [DOI] [PubMed] [Google Scholar]
  46. Yamada M., Miyawaki A., Saito K., Nakajima T., Yamamoto-Hino M., Ryo Y., Furuichi T., Mikoshiba K. The calmodulin-binding domain in the mouse type 1 inositol 1,4,5-trisphosphate receptor. Biochem J. 1995 May 15;308(Pt 1):83–88. doi: 10.1042/bj3080083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yoneshima H., Miyawaki A., Michikawa T., Furuichi T., Mikoshiba K. Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors. Biochem J. 1997 Mar 1;322(Pt 2):591–596. doi: 10.1042/bj3220591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Yoshikawa F., Morita M., Monkawa T., Michikawa T., Furuichi T., Mikoshiba K. Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1996 Jul 26;271(30):18277–18284. doi: 10.1074/jbc.271.30.18277. [DOI] [PubMed] [Google Scholar]

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