Calcium Flux Assay in Xenopus Oocytes

Philip M. Murphy1

1 National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.20
DOI:  10.1002/0471142301.ns0420s11
Online Posting Date:  May, 2001
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Abstract

Many G protein‐coupled receptors of interest to neuroscientists induce transient increases in [Ca2+]i, which can be used as a convenient measure of receptor activation in a variety of applications. This unit describes a simple calcium flux assay applied to Xenopus oocytes

     
 
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Table of Contents

  • Reagents and Solutioins
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Xenopus oocytes microinjected with appropriate natural RNA or cRNA (unit 4.3)
  • ND96 solution (see recipe)
  • OR‐2 solution (see recipe)
  • 45CaCl 2 (25 mCi/mg; NEN Life Sciences)
  • Scintillation fluid (e.g., Ecoscint, National Diagnostics)
  • Agonist of interest
  • 1% (w/v) SDS solution
  • 2‐ml pipet with thumb‐controlled suction
  • Dissecting microscope
  • 96‐well flat‐bottom and round‐bottom polystyrene plates
  • Multichannel pipetter
  • 20‐ml scintillation vials or 96‐well plate for plate reader
  • β scintillation counter: standard or 96‐well plate format (e.g., TopCount, Packard Instruments)
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Figures

Videos

Literature Cited

Literature Cited
   Berridge, M.J. 1995. Inositol trisphosphate and calcium signaling. Ann. N.Y. Acad. Sci. 766:31‐43.
   Bootman, M.D. and Berridge, M.J. 1995. The elemental principles of calcium signaling. Cell 83:675‐678.
   Boton, R., Dascal, N., Gillo, B., and Lass, Y. 1989. Two calcium‐activated chloride conductances in Xenopus laevis oocytes permeabilized with the ionophore A23187. J. Physiol. (Lond.) 408:511‐534.
   Burg, M., Raffetseder, U., Grove, M., Klos, A., Kohl, J., and Bautsch, W. 1995. G alpha‐16 complements the signal transduction cascade of chemotactic receptors for complement factor C5a (C5a‐R) and N‐formylated peptides (fMLF‐R) in Xenopus laevis oocytes: G alpha‐16 couples to chemotactic receptors in Xenopus oocytes. FEBS Lett . 377:426‐428.
   Colman, A. 1984. Translation of eukaryotic messenger RNA in Xenopus oocytes In Transcription and Translation: A Practical Approach (B.D. Hames and S.J. Higgins, eds.) pp. 271‐301. IRL Press Ltd., Oxford.
   Giladi, E. and Spindel, E.R. 1991. Simple luminometric assay to detect phosphoinositol‐linked receptor expression in Xenopus oocytes. Biotechniques 10:744‐747.
   Grynkiewicz, G., Poenie, M., and Tsien, , R.Y. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440‐3450.
   Murphy, P.M. 1997. Calcium flux assay of chemokine receptor expression in Xenopus oocytes. Methods Enzymol. 288:108‐117.
   Murphy, P.M. and McDermott, D. 1991. Expression of the human formyl peptide receptor in Xenopus oocytes requires a complementary human factor. J. Biol. Chem. 266:12560‐12567.
   Murphy, P.M. and McDermott, D. 1992. The guanine nucleotide binding protein Gs activates a novel calcium transporter in Xenopus oocytes. J. Biol. Chem. 267:883‐888.
   Murphy, P.M. and Tiffany, H.L. 1991. Cloning of complementary DNA encoding a functional human interleukin‐8 receptor. Science 253:1280‐1283.
   Murphy, P.M., Gallin, E.K., and Tiffany, H.L. 1990a. Characterization of phagocytic cell receptors for C5a and platelet activating factor expressed in Xenopus oocytes. J. Immunol. 145:2227‐2233.
   Murphy, P.M., Gallin, E.K., Tiffany, H.L., and Malech, H.L. 1990b. The formyl peptide chemoattractant receptor is encoded by a 2 kilobase messenger RNA: Expression in Xenopus oocytes. FEBS Lett. 261:353‐357.
   Oron, Y., Dascal, N., Nadler, E., and Lupu, M. 1985. Inositol 1,4,5‐trisphosphate mimics muscarinic response in Xenopus oocytes. Nature 313:141‐143.
   Thomas, K.M., Pyun, H.Y., and Navarro, J. 1990. Molecular cloning of the fMet‐Leu‐Phe receptor from neutrophils. J. Biol. Chem. 265:20061‐20064.
   Vu, T.K., Hung, D.T., Wheaton, V.I., and Coughlin, S.R. 1991. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64:1057‐1068.
   Williams, J.A., McChesney, D.J., Calayag, M.C., Lingappa, V.R., and Logsdon, C.D. 1988. Expression of receptors for cholecystokinin and other Ca2+‐mobilizing hormones in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 85:4939‐4943.
Key References
   Bootman and Berridge, 1995. See above
  This is an outstanding review of calcium signaling in cells.
   Murphy and Tiffany, 1991. See above.
  This paper gives the first examples of the use of the calcium release assay in oocytes to identify the ligand for a candidate G protein–coupled receptor, the chemokine receptor CXCR2.
   Vu, et al., 1991. See above.
  This paper details the first use of the calcium release assay in functional expression cloning of a G protein–coupled receptor, the thrombin receptor PAR1.
   Williams, et al., 1988. See above.
  This paper describes the first calcium release assay applied to Xenopus oocytes.
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