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. 2002 Aug 1;365(Pt 3):659–667. doi: 10.1042/BJ20011789

The high-affinity calcium[bond]calmodulin-binding site does not play a role in the modulation of type 1 inositol 1,4,5-trisphosphate receptor function by calcium and calmodulin.

Elena Nosyreva 1, Tomoya Miyakawa 1, Zhengnan Wang 1, Lyuba Glouchankova 1, Akiko Mizushima 1, Masamitsu Iino 1, Ilya Bezprozvanny 1
PMCID: PMC1222719  PMID: 11972451

Abstract

Modulation of the inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)R) by cytosolic calcium (Ca(2+)) plays an essential role in Ca(2+) signalling, but structural determinants and mechanisms responsible for the InsP(3)R regulation by Ca(2+) are poorly understood. In the present study, we expressed rat InsP(3)R type 1 (InsP(3)R1) in Spodoptera frugiperda cells using a baculovirus-expression system and reconstituted the recombinant InsP(3)R1 into planar lipid bilayers for functional analysis. We observed only minor effects of 0.5 mM of calmodulin (CaM) antagonist W-7 on the Ca(2+) dependence of InsP(3)R1. Based on a previous analysis of mouse InsP(3)R1 [Yamada, Miyawaki, Saito, Nakajima, Yamamoto-Hino, Ryo, Furuichi and Mikoshiba (1995) Biochem J. 308, 83-88], we generated the Trp(1577)-->Ala (W1577A) mutant of rat InsP(3)R1 which lacks the high-affinity Ca(2+)[bond]CaM-binding site. We found that the W1577A mutant displayed a bell-shaped Ca(2+) dependence similar to the wild-type InsP(3)R1 in planar lipid bilayers. Activation of B cell receptors resulted in identical Ca(2+) signals in intact DT40 cells lacking the endogenous InsP(3)R and transfected with the wild-type InsP(3)R1 or the W1577A mutant cDNA subcloned into a mammalian expression vector. In the planar lipid bilayer experiments, we showed that both wild-type InsP(3)R1 and W1577A mutant were equally sensitive to inhibition by exogenous CaM. From these results, we concluded that the interaction of CaM with the high-affinity Ca(2+)[bond]CaM-binding site in the coupling domain of the InsP(3)R1 does not play a direct role in biphasic modulation of InsP(3)R1 by cytosolic Ca(2+) or in InsP(3)R1 inhibition by CaM.

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Selected References

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  1. Adkins C. E., Morris S. A., De Smedt H., Sienaert I., Török K., Taylor C. W. Ca2+-calmodulin inhibits Ca2+ release mediated by type-1, -2 and -3 inositol trisphosphate receptors. Biochem J. 2000 Jan 15;345(Pt 2):357–363. [PMC free article] [PubMed] [Google Scholar]
  2. Asano M. Divergent pharmacological effects of three calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), chlorpromazine and calmidazolium, on isometric tension development and myosin light chain phosphorylation in intact bovine tracheal smooth muscle. J Pharmacol Exp Ther. 1989 Nov;251(2):764–773. [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. doi: 10.1038/361315a0. [DOI] [PubMed] [Google Scholar]
  4. Bezprozvanny I., Ehrlich B. E. ATP modulates the function of inositol 1,4,5-trisphosphate-gated channels at two sites. Neuron. 1993 Jun;10(6):1175–1184. doi: 10.1016/0896-6273(93)90065-y. [DOI] [PubMed] [Google Scholar]
  5. Bezprozvanny I., Ehrlich B. E. Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium. J Gen Physiol. 1994 Nov;104(5):821–856. doi: 10.1085/jgp.104.5.821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bezprozvanny I., Ehrlich B. E. The inositol 1,4,5-trisphosphate (InsP3) receptor. J Membr Biol. 1995 Jun;145(3):205–216. doi: 10.1007/BF00232713. [DOI] [PubMed] [Google Scholar]
  7. Bezprozvanny I., Watras J., Ehrlich B. E. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature. 1991 Jun 27;351(6329):751–754. doi: 10.1038/351751a0. [DOI] [PubMed] [Google Scholar]
  8. Boehning D., Joseph S. K. Functional properties of recombinant type I and type III inositol 1, 4,5-trisphosphate receptor isoforms expressed in COS-7 cells. J Biol Chem. 2000 Jul 14;275(28):21492–21499. doi: 10.1074/jbc.M001724200. [DOI] [PubMed] [Google Scholar]
  9. Cardy T. J., Taylor C. W. A novel role for calmodulin: Ca2+-independent inhibition of type-1 inositol trisphosphate receptors. Biochem J. 1998 Sep 1;334(Pt 2):447–455. doi: 10.1042/bj3340447. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. De Young G. W., Keizer J. A single-pool inositol 1,4,5-trisphosphate-receptor-based model for agonist-stimulated oscillations in Ca2+ concentration. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9895–9899. doi: 10.1073/pnas.89.20.9895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  13. Finch E. A., Turner T. J., Goldin S. M. Calcium as a coagonist of inositol 1,4,5-trisphosphate-induced calcium release. Science. 1991 Apr 19;252(5004):443–446. doi: 10.1126/science.2017683. [DOI] [PubMed] [Google Scholar]
  14. Furuichi T., Kohda K., Miyawaki A., Mikoshiba K. Intracellular channels. Curr Opin Neurobiol. 1994 Jun;4(3):294–303. doi: 10.1016/0959-4388(94)90089-2. [DOI] [PubMed] [Google Scholar]
  15. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  16. Hagar R. E., Burgstahler A. D., Nathanson M. H., Ehrlich B. E. Type III InsP3 receptor channel stays open in the presence of increased calcium. Nature. 1998 Nov 5;396(6706):81–84. doi: 10.1038/23954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hidaka H., Sasaki Y., Tanaka T., Endo T., Ohno S., Fujii Y., Nagata T. N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, a calmodulin antagonist, inhibits cell proliferation. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4354–4357. doi: 10.1073/pnas.78.7.4354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hirota J., Michikawa T., Natsume T., Furuichi T., Mikoshiba K. Calmodulin inhibits inositol 1,4,5-trisphosphate-induced calcium release through the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1. FEBS Lett. 1999 Aug 6;456(2):322–326. doi: 10.1016/s0014-5793(99)00973-4. [DOI] [PubMed] [Google Scholar]
  19. Horn R. Estimating the number of channels in patch recordings. Biophys J. 1991 Aug;60(2):433–439. doi: 10.1016/S0006-3495(91)82069-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iino M. Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci. J Gen Physiol. 1990 Jun;95(6):1103–1122. doi: 10.1085/jgp.95.6.1103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Islam M. O., Yoshida Y., Koga T., Kojima M., Kangawa K., Imai S. Isolation and characterization of vascular smooth muscle inositol 1,4,5-trisphosphate receptor. Biochem J. 1996 May 15;316(Pt 1):295–302. doi: 10.1042/bj3160295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kaftan E. J., Ehrlich B. E., Watras J. Inositol 1,4,5-trisphosphate (InsP3) and calcium interact to increase the dynamic range of InsP3 receptor-dependent calcium signaling. J Gen Physiol. 1997 Nov;110(5):529–538. doi: 10.1085/jgp.110.5.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaznacheyeva E., Lupu V. D., Bezprozvanny I. Single-channel properties of inositol (1,4,5)-trisphosphate receptor heterologously expressed in HEK-293 cells. J Gen Physiol. 1998 Jun;111(6):847–856. doi: 10.1085/jgp.111.6.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lechleiter J. D., Clapham D. E. Molecular mechanisms of intracellular calcium excitability in X. laevis oocytes. Cell. 1992 Apr 17;69(2):283–294. doi: 10.1016/0092-8674(92)90409-6. [DOI] [PubMed] [Google Scholar]
  25. Lin C., Widjaja J., Joseph S. K. The interaction of calmodulin with alternatively spliced isoforms of the type-I inositol trisphosphate receptor. J Biol Chem. 2000 Jan 28;275(4):2305–2311. doi: 10.1074/jbc.275.4.2305. [DOI] [PubMed] [Google Scholar]
  26. Mak D. O., McBride S., Foskett J. K. Inositol 1,4,5-trisphosphate [correction of tris-phosphate] activation of inositol trisphosphate [correction of tris-phosphate] receptor Ca2+ channel by ligand tuning of Ca2+ inhibition. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15821–15825. doi: 10.1073/pnas.95.26.15821. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mak D. O., McBride S., Foskett J. K. Regulation by Ca2+ and inositol 1,4,5-trisphosphate (InsP3) of single recombinant type 3 InsP3 receptor channels. Ca2+ activation uniquely distinguishes types 1 and 3 insp3 receptors. J Gen Physiol. 2001 May;117(5):435–446. doi: 10.1085/jgp.117.5.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Michikawa T., Hirota J., Kawano S., Hiraoka M., Yamada M., Furuichi T., Mikoshiba K. Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor. Neuron. 1999 Aug;23(4):799–808. doi: 10.1016/s0896-6273(01)80037-4. [DOI] [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. Missiaen L., DeSmedt H., Bultynck G., Vanlingen S., Desmet P., Callewaert G., Parys J. B. Calmodulin increases the sensitivity of type 3 inositol-1,4, 5-trisphosphate receptors to Ca(2+) inhibition in human bronchial mucosal cells. Mol Pharmacol. 2000 Mar;57(3):564–567. doi: 10.1124/mol.57.3.564. [DOI] [PubMed] [Google Scholar]
  31. Missiaen L., Parys J. B., Sienaert I., Maes K., Kunzelmann K., Takahashi M., Tanzawa K., De Smedt H. Functional properties of the type-3 InsP3 receptor in 16HBE14o- bronchial mucosal cells. J Biol Chem. 1998 Apr 10;273(15):8983–8986. doi: 10.1074/jbc.273.15.8983. [DOI] [PubMed] [Google Scholar]
  32. Missiaen L., Parys J. B., Weidema A. F., Sipma H., Vanlingen S., De Smet P., Callewaert G., De Smedt H. The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin. J Biol Chem. 1999 May 14;274(20):13748–13751. doi: 10.1074/jbc.274.20.13748. [DOI] [PubMed] [Google Scholar]
  33. Miyakawa T., Maeda A., Yamazawa T., Hirose K., Kurosaki T., Iino M. Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes. EMBO J. 1999 Mar 1;18(5):1303–1308. doi: 10.1093/emboj/18.5.1303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miyakawa T., Mizushima A., Hirose K., Yamazawa T., Bezprozvanny I., Kurosaki T., Iino M. Ca(2+)-sensor region of IP(3) receptor controls intracellular Ca(2+) signaling. EMBO J. 2001 Apr 2;20(7):1674–1680. doi: 10.1093/emboj/20.7.1674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Parker I., Choi J., Yao Y. Elementary events of InsP3-induced Ca2+ liberation in Xenopus oocytes: hot spots, puffs and blips. Cell Calcium. 1996 Aug;20(2):105–121. doi: 10.1016/s0143-4160(96)90100-1. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Ramos-Franco J., Caenepeel S., Fill M., Mignery G. Single channel function of recombinant type-1 inositol 1,4,5-trisphosphate receptor ligand binding domain splice variants. Biophys J. 1998 Dec;75(6):2783–2793. doi: 10.1016/S0006-3495(98)77721-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ramos-Franco J., Fill M., Mignery G. A. Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels. Biophys J. 1998 Aug;75(2):834–839. doi: 10.1016/S0006-3495(98)77572-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sipma H., De Smet P., Sienaert I., Vanlingen S., Missiaen L., Parys J. B., De Smedt H. Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin. J Biol Chem. 1999 Apr 23;274(17):12157–12162. doi: 10.1074/jbc.274.17.12157. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Swatton J. E., Morris S. A., Cardy T. J., Taylor C. W. Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization. Biochem J. 1999 Nov 15;344(Pt 1):55–60. [PMC free article] [PubMed] [Google Scholar]
  43. Taylor C. W. Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+ channels. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):19–33. doi: 10.1016/s0005-2760(98)00122-2. [DOI] [PubMed] [Google Scholar]
  44. Tu Huiping, Miyakawa Tomoya, Wang Zhengnan, Glouchankova Lyuba, Iino Masamitsu, Bezprozvanny Ilya. Functional characterization of the type 1 inositol 1,4,5-trisphosphate receptor coupling domain SII(+/-) splice variants and the Opisthotonos mutant form. Biophys J. 2002 Apr;82(4):1995–2004. doi: 10.1016/S0006-3495(02)75548-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vanlingen S., Sipma H., De Smet P., Callewaert G., Missiaen L., De Smedt H., Parys J. B. Ca2+ and calmodulin differentially modulate myo-inositol 1,4, 5-trisphosphate (IP3)-binding to the recombinant ligand-binding domains of the various IP3 receptor isoforms. Biochem J. 2000 Mar 1;346(Pt 2):275–280. [PMC free article] [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]

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