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

Scott Perschke1, John Ferkany2

1 NovaScreen Bioscience Corporation, Hanover, Maryland, 2 Virtual Drug Discovery, Inc., Avon, Connecticut
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.27
DOI:  10.1002/0471141755.ph0127s17
Online Posting Date:  August, 2002
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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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Table of Contents

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

  • 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 [3H]MDL‐105,519 (50 to 90 Ci/mmol; Amersham) or 100 nM [3H]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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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.
   Cheng, Y. and Prusoff, W.H. 1973. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3108.
   Conn, P.J. and Pin, J.‐P. 1997. Pharmacology and functions of metabotropic glutamate receptors. Ann. Rev. Pharmacol. Toxicol. 37:205‐237.
   Evans, R.H. and Watkins, J.C. 1981. Pharmacological antagonists of excitant amino acid action. Life Sci. 28:1303‐1308.
   Fischer, G., Mutel, V., Trube, G., Malherbe, P., Kew, J.N., Mohacsi, E., Heitz, M.P., and Kemp, J.A. 1997. Ro 25‐6981, a highly potent and selective blocker of N‐methyl‐D‐aspartate receptors containing the NR2B subunit: Characterization in vitro. J. Pharmacol. Exp. Ther. 283:1285‐1292.
   Fisher, K., Coderre, T.J. and Hagen, N.A. 2000. Targeting the N‐methyl‐D‐aspartate receptor for chronic pain management. Preclinical animal studies, recent clinical experience and future research directions. J. Pain Symptom. Manage 20:358‐373.
   Grimwood, S., Richards, P., Murray, F., Harrison, N., Wingrove, P.B. and Hutson, P.H. 2000. Characterisation of N‐methyl‐D‐aspartate receptor‐specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes. J. Neurochem. 75:2455‐2463.
   Ivanovic, A., Reilander, H., Laube, B. and Kuhse, J. 1998. Expression and initial characterization of a soluble glycine binding domain of the N‐methyl‐D‐aspartate receptor NR1 subunit. J. Biol. Chem. 273:19933‐19937.
   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.
   Nakanishi, M. and Masu, M. 1994. Molecular diversity and functions of glutamate receptors. Ann. Rev. Biophys. Biomol. Struct. 23:319‐348.
   Petrie, R.X., Reid, I.C., and Stewart, C.A. 2000. The N‐methyl‐D‐aspartate receptor, synaptic plasticity, and depressive disorder: A critical review. Pharmacol. Ther. 87:11‐25.
   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.
   Snyder, S.H. and Kim, P.M. 2000. D‐amino acids as putative neurotransmitters: Focus on D‐serine. Neurochem. Res. 25:553‐560.
   Young, A.B., and Snyder, S.H. 1974. Strychnine binding in rat spinal cord membranes associated with the synaptic glycine receptors: cooperativity of glycine interactions. Mol. Pharmacol. 10:790‐809.
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.
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