Radioligand Binding Assays for the Glycine Site on N‐Methyl‐D‐Aspartate Receptors

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes a competitive binding assay for the glycine binding site on the NMDA subtype of glutamate receptors in rat brain homogenates. Agonists of the NMDA receptor associated glycine binding site have been proposed as potential therapeutics in cognitive disorders. Conversely, antagonists may be useful in a variety of disorders associated with excessive activation of EAA receptors, including Parkinsons, Huntingtons and Alzheimers Diseases, and neuropathic pain, among others. This unit describes a competitive binding assay for the glycine binding site on the NMDA subtype of glutamate receptors in rat

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Basic Protocol 1: Measurement of Radioligand Binding to the Glycine Site on the NMDA Receptor
Commentary
Figures
Tables

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Materials

Basic Protocol 1: Measurement of Radioligand Binding to the Glycine Site on the NMDA Receptor

Materials

Male Sprague‐Dawley rats (125 to 200 g)
0.32 M sucrose, ice‐cold
High‐quality deionized and pyrogen‐free water (filter deionized water through 0.2‐µm filter)
50 mM Tris‐acetate, pH 7.4 at room temperature (adjust pH with acetic acid or Tris base), chilled to ice‐cold (pH will be ∼8.0 at 4°C)
Test compounds at 10× final concentration, and vehicle controls (DMSO or ethanol up to final concentration of 5% in assay mixture can be used as vehicle)
Compounds for nonspecific binding determination at 10× final concentration: 1 mM (10×) glycine (prepare fresh) or 30 µM (10×) MDL‐105,519 (Sigma), store in aliquots up to 6 months at –40°C
10× Radioligand: 40 nM [³ H]MDL‐105,519 (50 to 90 Ci/mmol; Amersham) or 100 nM [³ H]glycine (30 to 60 Ci/mmol; NEN/Perkin Elmer or Amersham)
0.1% (v/v) polyethyleneimine (PEI; Sigma), ice‐cold
50 mM NaCl, ice‐cold

Potter‐Elvehjem homogenizer with motor‐driven Teflon pestle (VWR), chilled to 4°C
50‐ml polypropylene centrifuge tubes
Refrigerated centrifuge and rotor capable of generating 50,000 × g
Polytron (Brinkmann) or equivalent tissue homogenizer
12 × 75–mm polypropylene or borosilicate glass tubes and rack, microtiter plates, or 1.2‐ml strip tubes
GF/C filter mats (Brandel) or filter plates (Packard)
Vacuum harvester (Brandel, or equivalent; unique harvester required for each tube format; LSM harvester for 48‐well, plate harvester for 96‐well)
Scintillation vials (if not using filter plates)
Aqueous‐compatible scintillation fluor (as recommended by the manufacturer of the counter)

NOTE : When stored at 4°C, the assay and wash buffers are stable for at least one week. If there is any evidence of bacterial or fungal contamination, discard the buffer immediately.

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Figures

Figure 1.27.1 Competition for sites labeled by [³ H]MDL‐105,519. Typical curves for competitors (agonist and antagonist) for the glycine site on the NMDA ion channel using [³ H]MDL‐105,519 as the radioligand. Abbreviations: 5,7‐DCKA, 5,7‐dichlorokynurenic acid; HA966, R‐(+)‐3‐amino‐1‐hydroxy‐2‐pyrrolidinone. All compounds mentioned here are available from Sigma.

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Figure 1.27.2 A representive curve of competition by glycine for sites labeled with [³ H]glycine. K _i values for other reference compounds in competition with [₃ H]glycine are shown in Table .

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Literature Cited

Literature Cited
	Baron, B.M., Harrison, B.L., Kehne, J.H., Schmidt, C.J., van Giersbergen, P.L., White, H.S., Siegel, B.W., Senyah, Y., McCloskey, T.C., Fadayel, G.M., Taylor, V.L., Murawsky, M.K., Nyce, P., and Salituro, F.G.. 1997. Pharmacological characterization of MDL‐105,519, an NMDA receptor glycine site antagonist. Eur. J. Pharmacol. 323:181‐192.
	Bisaga, A., Popik, P., Bespalov, A.Y., and Danysz, W. 2000. Therapeutic potential of NMDA receptor antagonists in the treatment of alcohol and substance use disorders. Expert Opin. Investig. Drugs. 9:2233‐2248.
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	Monahan, J.B., Corpus, V.M., Hood, W.F., Thomas, J.W. and Compton, R.P. 1989. Characterization of a [3H]‐glycine recognition site as a modulatory site of the NMDA receptor complex. J. Neurochem. 53:370‐375.
	Munoz, L.M., DeFeudis, F.V., and Fando, J.L. 1997. High‐affinity binding of glycine and beta‐alanine to synaptosome‐enriched fractions of rat CNS regions: Effects of strychnine. Gen. Pharmacol. 8:325‐330.
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	Ruiz‐Gomez, A., Garcia‐Calvo, M., Vazquez, J., Marvizon, J.C., Valdivieso, F. and Mayor, F. Jr. 1989. Thermodynamics of agonist and antagonist interaction with the strychnine‐sensitive glycine receptor. J. Neurochem. 52:1775‐1780.
	Siegel, B.W., Sreekrishna, K., and Baron, B.M., 1996. Binding of the radiolabeled glycine site antagonist [3H]MDL‐105,519 to homomeric NMDA‐NR1a receptors. Eur. J. Pharmacol. 312:357‐365.
	Snell, L.D., Morter, R.S., and Johnson, K.M. 1988. Structural requirements for activation of the glycine receptor that modulates the N‐methyl‐D‐aspartate operated channel. Eur. J. Pharmacol. 156:105‐110.
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Key References
	Baron et al., 1997. See above.
	The primary article describing the characterization of [3H]MDL‐105,519 binding to the glycine site.
	Johnson, J.W. and Ascher, P. 1987. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529‐531.
	The original work demonstrating the specific interaction of glycine with the NMDA channel.