[3H](+)MK801 Radioligand Binding Assay at the N‐Methyl‐D‐Aspartate Receptor

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

The N ?methyl?D ?aspartate subtype of glutamate receptor is unusual in that it requires two endogenous agonists for activation. Thus, in addition to glutamate, the amino acid glycine (or possibly D ?serine) is an essential co?agonist. This unit presents a radioligand binding protocol that detects ligand activity at the NMDA receptor?associated glycine site. This is a convenient approach that exploits the ability of NMDA receptor modulators to alter the kinetics of ligands that bind to the channel?blocking site of the NMDA receptor. This protocol takes advantage of one of the most potent and specific ligands that bind to this receptor, in this case [³ H](+)MK801. Importantly, this assay can detect and differentiate agonists and antagonists that bind to the glycine site. A protocol for the measurement of glycine site activity with [³ H](+)MK801 binding is provided, along with support protocols that provide information to aid in the design of assays of agonists and antagonists of the glycine site, and data analysis.

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Basic Protocol 1: Measurment of NMDA Receptor‐Associated Glycine Site Activity with [3H](+)MK801 Binding
Alternate Protocol 1: Assaying Glycine Site Agonists and Antagonists
Support Protocol 1: Data Analysis
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Measurment of NMDA Receptor‐Associated Glycine Site Activity with [3H](+)MK801 Binding

Materials

Rat brains (may be fresh or frozen)
20 mM HEPES, pH 7.4, containing 1 mM tetrasodium EDTA, room temperature and 4°C
20 mM HEPES, pH 7.4, room temperature and 4°C
22 to 25 Ci/mmol [³ H](+)MK801 (NEN Life Sciences)
1 mM glutamic acid in deionized water (stable for > month at −20°C)
Test compounds
Water compatible scintillation cocktail (e.g., ScintiSafe 30%, Fisher Scientific)

50‐ml polycarbonate centrifuge tubes
Polytron homogenizer (Brinkmann)
Glass fiber filter strips (e.g., Whatman GF‐B) compatible with cell harvester
Scintillation vials

Additional reagents and equipment for total protein assays ( appendix 3A )

NOTE: HEPES buffer is stable up to 1 month at room temperature, but visually inspect the solution periodically to ensure that there is no growth of bacteria or fungi which may be a source of contaminating glycine. This is not typically a problem with deionized or distilled water sources.

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Figures

Figure 1.20.1 Typical curves for agonist and antagonist compounds. (A ) These curves show the anticipated profile of a glycine site agonist added alone (open symbols) or in the presence (filled symbols) of the glycine site antagonist, 5,7‐dichlorokynurenate (5 µM). Data are modeled based on the expected results from the protocol provided here. Note that the addition of the antagonist reduces basal binding. This results from the inhibition of the binding of the endogenous glycine in the membrane preparation. Note also that the EC₅₀ value of the test compound will shift to the right in the presence of 5,7‐dichlorokynurenate, consistent with a competitive interaction of the test compound with the glycine site. The EC₅₀ value of the test compound in this case is 2 µM. (B ) These curves show the anticipated profile of a glycine site antagonist added in the nominal absence (open symbols) or presence (filled symbols) of glycine (10 µM). The addition of glycine increases the amount of binding and also shifts the IC₅₀ value to the right, again consistent with a competitive interaction with the glycine site. The IC₅₀ value of the antagonist in this example is 0.1 µM. Total binding is shown in these graphs, and the nonspecific binding is 500 cpm.

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Literature Cited

	Baron, B.M., Siegel, B.W., Harrison, B.L., Gross, R.S., Hawes, C., and Towers, P. 1996. [3H]MDL 105,519, a high‐affinity radioligand for the N‐methyl‐D‐aspartate receptor‐associated glycine recognition site J. Pharmacol. Exp. Ther. 279:62‐68.
	Bonhaus, D.W. and McNamara, J.O. 1998. N‐methyl‐D‐aspartate receptor regulation of uncompetitive antagonist binding in rat brain membranes: Kinetic analysis. Mol. Pharmacol. 34:250‐255.
	Grimwood, S., Moseley, A.M., Carling, R.W., Leeson, P.D. and Foster, A.C. 1992. Characterization of the binding of [3H]L‐689,560, an antagonist for the glycine site on the N‐methyl‐D‐aspartate receptor, to rat brain membranes. Mol. Pharmacol. 41:923‐930.
	Johnson, J.W. and Ascher, P. 1987. Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529‐531.
	Kemp, J.A. and Leeson, P.D. 1993. The glycine site of the NMDA receptor–Five years on. Trends Pharmacol. Sci. 14:20‐25.
	Kleckner, N.W. and Dingledine, R. 1988. Requirement for glycine in activation of NMDA receptors expressed in Xenopus oocytes. Science 241:835‐837.
	Kloog, Y., Nadler, V., and Sokolovsky, M. 1998. Mode of binding of [3H]dibenzocycloalkenimine (MK‐801) to the N‐methyl‐D‐aspartate (NMDA) receptor and its therapeutic implication. FEBS Lett. 230:167‐170.
	Leeson, P.D. and Iversen, L.L. 1994. The glycine site on the NMDA receptor: Structure‐activity relationships and therapeutic potential. J. Med. Chem. 37:4053‐4067.
	McBain, C.J. and Mayer, M.L. 1994. N‐methyl‐D‐aspartic acid receptor structure and function. Physiol. Rev. 74:723‐760.
	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 N‐methyl‐D‐aspartate receptor complex. J. Neurochem. 53:370‐375.
	Rajdev, S. and Reynolds, I.J. 1993. Effects of pH on the actions of dizocilpine at the N‐methyl‐D‐aspartate receptor complex. Br. J. Pharmacol. 109:107‐112.
	Reynolds, I.J., Murphy, S.N., and Miller, R.J. 1987. 3H‐labelled MK‐801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glycine. Proc. Natl. Acad. Sci. U.S.A. 84:7744‐7748.
	Reynolds, I.J. and Palmer, A.M. 1991. Regional variations in [3H]MK801 binding to rat brain NMDA receptors. J. Neurochem. 56:1731‐1740.
	Reynolds, I.J. and Rothermund, K.D. 1995. Characterization of the effects of polyamines on the modulation of the N‐methyl‐D‐aspartate receptor by glycine. Neuropharmacol. 34:1147‐1157.
	Starmer, C.F., Packer, D.L., and Grant, A.O. 1987. Ligand binding to transiently accessible sites: Mechanisms for varying apparent binding rates. J. Theoret. Biol. 124:335‐341.
	Wolosker, H., Blackshaw, S., and Snyder, S.H. 1999. Serine racemase: A glial enzyme synthesizing D‐serine to regulate glutamate‐N‐methyl‐D‐aspartate neurotransmission. Natl. Acad. Sci. U.S.A. 96:13409‐13414.
Key References
	Johnson, J.W. and Ascher, P. 1987, See above.
	First demonstration of glycine modulation of the NMDA receptor.

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[3H](+)MK801 Radioligand Binding Assay at the N‐Methyl‐D‐Aspartate Receptor

Abstract

Table of Contents

Materials

Basic Protocol 1: Measurment of NMDA Receptor‐Associated Glycine Site Activity with [3H](+)MK801 Binding

Figures

Videos

Literature Cited