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. 1997 Jul 1;325(Pt 1):177–182. doi: 10.1042/bj3250177

Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar microsomes.

M Mezna 1, F Michelangeli 1
PMCID: PMC1218543  PMID: 9224644

Abstract

Thimerosal, a thiol-reactive reagent, has been shown to increase the cytosolic Ca2+ concentration in a variety of cells by sensitizing inositol 1,4,5-trisphosphate (InsP3) receptors. Thimerosal can have both sensitizing (at concentrations of <2 microM) and inhibitory (at concentrations of >2 microM) effects on InsP3-induced Ca2+ release (IICR) from cerebellar microsomes. Transient kinetic studies were performed by employing a fluorimetric stopped-flow approach using fluo-3. IICR was found to be a bi-exponential process with a fast and a slow component. At a maximal InsP3 concentration (20 microM), the fast phase had a rate constant of 0.9 s-1 and the slow phase had a rate constant of 0.4 s-1. The amplitudes of the two phases were 60% and 40% respectively. When the rate constants for the two phases were plotted as Hill plots, the processes were found to be non-co-operative in both cases (Hill coefficient of 1.0), thus arguing for a simple mechanism linking InsP3 binding to channel opening. At a submaximal InsP3 concentration (0.2 microM), where the sensitizing effects of thimerosal are most pronounced, thimerosal increased the rate constants of both phases in a sigmoidal fashion, with a Hill coefficient of 4.0, suggesting that several cysteine residues (up to four) need to be modified in order for maximum sensitization to occur. The rate constants remained elevated even at thimerosal concentrations that inhibited IICR. The amplitude or extent of Ca2+ release was, however, elevated to a much greater extent in the slow phase, suggesting that the two phases respond differently. At maximal InsP3 concentrations, thimerosal has no effect upon the rate constants but inhibits the amplitude of Ca2+ release.

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Selected References

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  1. Abramson J. J., Zable A. C., Favero T. G., Salama G. Thimerosal interacts with the Ca2+ release channel ryanodine receptor from skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1995 Dec 15;270(50):29644–29647. doi: 10.1074/jbc.270.50.29644. [DOI] [PubMed] [Google Scholar]
  2. Bootman M. D., Taylor C. W., Berridge M. J. The thiol reagent, thimerosal, evokes Ca2+ spikes in HeLa cells by sensitizing the inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1992 Dec 15;267(35):25113–25119. [PubMed] [Google Scholar]
  3. Champeil P., Combettes L., Berthon B., Doucet E., Orlowski S., Claret M. Fast kinetics of calcium release induced by myo-inositol trisphosphate in permeabilized rat hepatocytes. J Biol Chem. 1989 Oct 25;264(30):17665–17673. [PubMed] [Google Scholar]
  4. Furuichi T., Yoshikawa S., Miyawaki A., Wada K., Maeda N., Mikoshiba K. Primary structure and functional expression of the inositol 1,4,5-trisphosphate-binding protein P400. Nature. 1989 Nov 2;342(6245):32–38. doi: 10.1038/342032a0. [DOI] [PubMed] [Google Scholar]
  5. Galione A., McDougall A., Busa W. B., Willmott N., Gillot I., Whitaker M. Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs. Science. 1993 Jul 16;261(5119):348–352. doi: 10.1126/science.8392748. [DOI] [PubMed] [Google Scholar]
  6. Genazzani A. A., Mezna M., Summerhill R. J., Galione A., Michelangeli F. Kinetic properties of nicotinic acid adenine dinucleotide phosphate-induced Ca2+ release. J Biol Chem. 1997 Mar 21;272(12):7669–7675. doi: 10.1074/jbc.272.12.7669. [DOI] [PubMed] [Google Scholar]
  7. Guillemette G., Segui J. A. Effects of pH, reducing and alkylating reagents on the binding and Ca2+ release activities of inositol 1,4,5-triphosphate in the bovine adrenal cortex. Mol Endocrinol. 1988 Dec;2(12):1249–1255. doi: 10.1210/mend-2-12-1249. [DOI] [PubMed] [Google Scholar]
  8. Hilly M., Piétri-Rouxel F., Coquil J. F., Guy M., Mauger J. P. Thiol reagents increase the affinity of the inositol 1,4,5-trisphosphate receptor. J Biol Chem. 1993 Aug 5;268(22):16488–16494. [PubMed] [Google Scholar]
  9. Hirota J., Michikawa T., Miyawaki A., Takahashi M., Tanzawa K., Okura I., Furuichi T., Mikoshiba K. Adenophostin-medicated quantal Ca2+ release in the purified and reconstituted inositol 1,4,5-trisphosphate receptor type 1. FEBS Lett. 1995 Jul 17;368(2):248–252. doi: 10.1016/0014-5793(95)00659-w. [DOI] [PubMed] [Google Scholar]
  10. Joseph S. K., Ryan S. V., Pierson S., Renard-Rooney D., Thomas A. P. The effect of mersalyl on inositol trisphosphate receptor binding and ion channel function. J Biol Chem. 1995 Feb 24;270(8):3588–3593. doi: 10.1074/jbc.270.8.3588. [DOI] [PubMed] [Google Scholar]
  11. Kaplin A. I., Ferris C. D., Voglmaier S. M., Snyder S. H. Purified reconstituted inositol 1,4,5-trisphosphate receptors. Thiol reagents act directly on receptor protein. J Biol Chem. 1994 Nov 18;269(46):28972–28978. [PubMed] [Google Scholar]
  12. Katayama E., Funahashi H., Michikawa T., Shiraishi T., Ikemoto T., Iino M., Mikoshiba K. Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy. EMBO J. 1996 Sep 16;15(18):4844–4851. [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Meyer T., Wensel T., Stryer L. Kinetics of calcium channel opening by inositol 1,4,5-trisphosphate. Biochemistry. 1990 Jan 9;29(1):32–37. doi: 10.1021/bi00453a004. [DOI] [PubMed] [Google Scholar]
  15. Mezna M., Michelangeli F. Alkali metal ion dependence of inositol 1,4,5-trisphosphate-induced calcium release from rat cerebellar microsomes. J Biol Chem. 1995 Nov 24;270(47):28097–28102. doi: 10.1074/jbc.270.47.28097. [DOI] [PubMed] [Google Scholar]
  16. Mezna M., Michelangeli F. Opening up Ca2+ stores with InsP3. Nature. 1995 Jul 27;376(6538):300–301. doi: 10.1038/376300a0. [DOI] [PubMed] [Google Scholar]
  17. Mezna M., Michelangeli F. The effects of inositol 1,4,5-trisphosphate (InsP3) analogues on the transient kinetics of Ca2+ release from cerebellar microsomes. InsP3 analogues act as partial agonists. J Biol Chem. 1996 Dec 13;271(50):31818–31823. doi: 10.1074/jbc.271.50.31818. [DOI] [PubMed] [Google Scholar]
  18. Mezna M., Patchick T., Tovey S., Michelangeli F. Inhibition of the cerebellar inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by ethanol and other aliphatic alcohols. Biochem J. 1996 Feb 15;314(Pt 1):175–179. doi: 10.1042/bj3140175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Michelangeli F., Mezna M., Tovey S., Sayers L. G. Pharmacological modulators of the inositol 1,4,5-trisphosphate receptor. Neuropharmacology. 1995 Sep;34(9):1111–1122. doi: 10.1016/0028-3908(95)00053-9. [DOI] [PubMed] [Google Scholar]
  20. Michelangeli F. The effects of amino acid-reactive reagents on the functioning of the inositol 1,4,5-trisphosphate-sensitive calcium channel from rat cerebellum. Cell Signal. 1993 Jan;5(1):33–39. doi: 10.1016/0898-6568(93)90005-7. [DOI] [PubMed] [Google Scholar]
  21. Missiaen L., Taylor C. W., Berridge M. J. Spontaneous calcium release from inositol trisphosphate-sensitive calcium stores. Nature. 1991 Jul 18;352(6332):241–244. doi: 10.1038/352241a0. [DOI] [PubMed] [Google Scholar]
  22. Miyazaki S., Shirakawa H., Nakada K., Honda Y., Yuzaki M., Nakade S., Mikoshiba K. Antibody to the inositol trisphosphate receptor blocks thimerosal-enhanced Ca(2+)-induced Ca2+ release and Ca2+ oscillations in hamster eggs. FEBS Lett. 1992 Sep 7;309(2):180–184. doi: 10.1016/0014-5793(92)81090-9. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Palade P., Dettbarn C., Alderson B., Volpe P. Pharmacologic differentiation between inositol-1,4,5-trisphosphate-induced Ca2+ release and Ca2+- or caffeine-induced Ca2+ release from intracellular membrane systems. Mol Pharmacol. 1989 Oct;36(4):673–680. [PubMed] [Google Scholar]
  25. Parys J. B., Missiaen L., De Smedt H., Droogmans G., Casteels R. Bell-shaped activation of inositol-1,4,5-trisphosphate-induced Ca2+ release by thimerosal in permeabilized A7r5 smooth-muscle cells. Pflugers Arch. 1993 Sep;424(5-6):516–522. doi: 10.1007/BF00374916. [DOI] [PubMed] [Google Scholar]
  26. Poitras M., Bernier S., Servant M., Richard D. E., Boulay G., Guillemette G. The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state. J Biol Chem. 1993 Nov 15;268(32):24078–24082. [PubMed] [Google Scholar]
  27. Renard D. C., Seitz M. B., Thomas A. P. Oxidized glutathione causes sensitization of calcium release to inositol 1,4,5-trisphosphate in permeabilized hepatocytes. Biochem J. 1992 Jun 1;284(Pt 2):507–512. doi: 10.1042/bj2840507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rooney T. A., Renard D. C., Sass E. J., Thomas A. P. Oscillatory cytosolic calcium waves independent of stimulated inositol 1,4,5-trisphosphate formation in hepatocytes. J Biol Chem. 1991 Jul 5;266(19):12272–12282. [PubMed] [Google Scholar]
  29. Sayers L. G., Brown G. R., Michell R. H., Michelangeli F. The effects of thimerosal on calcium uptake and inositol 1,4,5-trisphosphate-induced calcium release in cerebellar microsomes. Biochem J. 1993 Feb 1;289(Pt 3):883–887. doi: 10.1042/bj2890883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thrower E. C., Duclohier H., Lea E. J., Molle G., Dawson A. P. The inositol 1,4,5-trisphosphate-gated Ca2+ channel: effect of the protein thiol reagent thimerosal on channel activity. Biochem J. 1996 Aug 15;318(Pt 1):61–66. doi: 10.1042/bj3180061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Watras J., Moraru I., Costa D. J., Kindman L. A. Two inositol 1,4,5-trisphosphate binding sites in rat basophilic leukemia cells: relationship between receptor occupancy and calcium release. Biochemistry. 1994 Nov 29;33(47):14359–14367. doi: 10.1021/bi00251a050. [DOI] [PubMed] [Google Scholar]
  32. 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]

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