User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

Calcium Flux Assay in Xenopus Oocytes

Philip M. Murphy¹

¹National 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

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Basic Protocol
Reagents and Solutioins
Commentary
Bibliography
Figures

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Materials

Basic Protocol

Materials

Xenopus oocytes microinjected with appropriate natural RNA or cRNA (unit 4.3)
ND96 solution (see recipe)
OR-2 solution (see recipe)
⁴⁵CaCl₂ (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
scintillation counter: standard or 96-well plate format (e.g., TopCount, Packard Instruments)

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 4.20.1

Time course for functional expression of chemoattractant receptors. Oocytes were injected with 50 ng of HL60 granulocyte RNA. The ⁴⁵Ca²⁺ release assay was performed on each day after injection using 100 nM fMet-Leu-Phe as the agonist. Data are means ± SEM of six oocytes per condition. Oocytes were loaded with ~1000 cpm on each day. Control oocytes injected with water (open circles) did not respond. For most purposes, the oocytes can be assayed on day 3 or 4, when activity peaks for most chemoattractant receptors studied thus far. Reprinted from Murphy (1997).

View Image
Figure 4.20.2

Kinetics of ⁴⁵Ca²⁺ release from oocytes expressing chemoattractant receptors. Oocytes injected with 50 ng of HL60 granulocyte RNA were loaded with ⁴⁵Ca²⁺. Each time point corresponds to the cpm present in a 100-µl wash incubated for the length of the interval shown. The arrow indicates the time of addition of 100 nM fMet-Leu-Phe (fMLF), which activates the N-formyl peptide receptor encoded by the transcripts in this natural RNA sample. Data are means ± SEM of six oocytes per condition. Oocytes contained ~10,000 cpm at time 0. Control oocytes injected with water (open circles) did not respond. For most purposes, the assay can be simplified by measuring cpm in the bath at 0 and 20 min, and by measuring the remaining cpm in the oocyte as described in the text. Reprinted from Murphy (1997).

View Image

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.