Characterization of Wild‐Type Excitatory Amino Acid Ion Channel Receptors

John W. Ferkany1

1 Oread, Inc., Farmington, Connecticut
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.14
DOI:  10.1002/0471141755.ph0114s04
Online Posting Date:  May, 2001
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Abstract

The ubiquitous distribution of EAA receptors results from the fact that EAA‐mediated neurotransmission is the most prevalent type of fast, chemically‐mediated communication in the brain and spinal cord. Indeed, EAA‐mediated neurotransmission is thought to participate in a host of physiological and pathological events including learning and memory, epilepsy, neurodegenerative conditions such as Alzhiemer's, Parkinson's and Huntington's Diseases, and the neurodegeneration associated with stroke and head trauma and neuropathic pain. Described in this unit are receptor binding assays for studying ion‐channel forming excitatory amino acid (EAA) receptors in the mammalian central nervous system. One of the three major types of ionotropic (channel‐forming) EAA receptors are the AMPA receptors, and the other two types are classified on the basis of the original agonists identified for these sites: kainic acid (kainate; KA) and N‐methyl‐D‐aspartic acid (NMDA). Thus, the three recognized ionotropic receptors are the AMPA, KA and NMDA recognition sites. Like GABA receptors, the AMPA and NMDA receptors have multiple regulatory sites.

     
 
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Table of Contents

  • Basic Protocol 1: Measurement of [3H]AMPA Binding to Wild‐Type AMPA Receptors
  • Alternate Protocol 1: Measurement of [3H]KA Binding to Wild‐Type KA Receptors
  • Alternate Protocol 2: Measurement of [3H]Ligand Binding to Wild‐Type NMDA Receptors
  • Support Protocol 1: Preparation of Membranes for Binding Assays
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of [3H]AMPA Binding to Wild‐Type AMPA Receptors

  Materials
  • Tris/thiocyanate buffer: 0.05 M Tris⋅Cl supplemented with 0.1 M potassium thiocyanate, pH 7.1 at 4°C
  • Buffy coat membrane suspension (see protocol 4)
  • L‐Glutamate (Sigma; RBI)
  • [3H](RS)‐α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid ([3H]AMPA; NEN Life Sciences)
  • Test compounds
  • Tissue solubilizer (e.g., Solvable; NEN Life Sciences; for centrifugation assays)
  • Scintillation cocktail (aqueous compatible; e.g., NEN Life Sciences)
  • 7‐ml plastic scintillation vials
  • Homogenizer (e.g., Polytron, Tekmar Tissumizer)
  • Centrifuge (Beckman J2‐21M with JA‐20 and JA‐20.1 rotors, or equivalent)
  • 12 × 75–mm borosilicate glass tubes or 1.5‐ml polypropylene tubes (for filtration assays)
  • Vacuum filtration tissue harvester (Brandell or equivalent) with pump (for filtration assays)
  • Glass fiber filters (Whatman GF/B or equivalent; for filtration assays)
CAUTION: Do not confuse potassium thiocyanate with potassium cyanide or potassium cyanate. The latter are extremely hazardous if not handled properly.

Alternate Protocol 1: Measurement of [3H]KA Binding to Wild‐Type KA Receptors

  • Tris buffer: 0.05 M Tris⋅Cl, pH 7.6 at 4°C ( appendix 2A)
  • [3H]Kainic acid ([3H]KA; NEN Life Sciences)
  • Kainic acid (Sigma; RBI)

Alternate Protocol 2: Measurement of [3H]Ligand Binding to Wild‐Type NMDA Receptors

  • Tris buffer: 0.05 M Tris⋅Cl, pH 7.6 at 4°C
  • 3H‐labeled radioligand (NEN Life Sciences): e.g., [3H]D‐(−)‐3‐(2‐carboxy‐piperazine‐ 4‐yl)propyl‐1‐phosphonic acid ([3H]CPP); [3H]‐(±)‐cis‐4‐phosphono‐methyl‐2‐piperidine carboxylic acid ([3H]CGS 19755); or [3H](±)‐E‐2‐amino‐4‐methyl‐5‐phosphono‐3‐pentanoic acid ([3H]CGP 37849)
  • N‐methyl‐D‐aspartate (NMDA), L‐glutamate, or CGS 19755 (Sigma; RBI)

Support Protocol 1: Preparation of Membranes for Binding Assays

  Materials
  • Experimental animals (e.g., rat, guinea pig, rabbit)
  • 0.32 M sucrose, 4°C
  • Small‐animal decapitator (or CO 2 chamber if required by IACUC)
  • Surgical instruments to penetrate cranium and remove forebrain
  • 20‐ to 40‐ml glass homogenizer vessel with Teflon pestle (use only manufacturer‐mated sets)
  • Homogenizer controller (∼700 rpm)
  • 40‐ to 50‐ml polypropylene centrifuge tubes
  • Beckman J2‐21M centrifuge with JA‐20 rotor (or equivalent)
  • Homogenizer (e.g., Polytron, Tekmar Tissumizer)
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Figures

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Literature Cited

Literature Cited
   Enna, S.J., and Snyder, S. 1976. Influences of ions, enzymes and detergents on gamma‐aminobutyric acid‐receptor binding in synaptic membranes of rat brain. Mol. Pharmacol. 13:442‐453.
   Ferkany, J.W., and Coyle, J.T. 1983. Specific binding of [3H](±)‐2‐amino‐7‐phosphono heptanoic acid to rat brain membranes. Life Sci. 33:1295‐1305.
   Kaplita, P.V., and Ferkany, J.W. 1990. Evidence for direct interactions between the NMDA and glycine recognition sites in brain. Eur. J. Pharmacol. 188:175‐179.
   Krogsgaard‐Larsen, P., Ebert, B., Lund, T.M., Brauner‐Osborne, H., Slok, F.A., Johansen, T.N., Brehm, L., and Madsen, U. 1996. Design of excitatory amino acid receptor agonists, partial agonists and antagonists: Ibotenic acid as a key lead structure. Eur. J. Med. Chem. 31:515‐537.
   Lehmann, J., Schneider, J., Mcpherson, S., Murphy, D.E., Bernard, P., Tsai, C., Bennett, D.A., Pastor, G., Steele, D.J., Boehm, C., Cheney, D.L., Liebmann, J.M., Williams, M., and Wood, P.L. 1987. CPP, a selective N‐methyl‐D‐aspartate (NMDA)‐type receptor antagonist: Characterization in vitro and in vivo. J. Pharmacol. Exp. Ther. 240:737‐746.
   London, E.D., and Coyle, J.T. 1979. Specific binding of [3H]kainic acid to receptor sites in rat brain. Mol. Pharmacol. 15:492‐505.
   Murphy, D.E., Snowhill, E.W., and Williams, M. 1987. Characterization of quisqualate recognition sites in rat brain tissue using DL‐[3H]α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) and a filtration assay. Neurochem. Res. 12:775‐781.
   Murphy, D.E., Hutchison, A.J., Hurt, S.D., Willams, M., and Sills, M.A. 1988. Characterization of the binding of [3H]CGS‐19755: A novel N‐methyl‐D‐aspartate antagonist with nanomolar affinity in rat brain. Br. J. Pharmacol. 95:932‐938.
   Sills, M.A., Fagg, G., Pozz, M., Angst, C., Brundish, D.E., Hurt, S.D., Wilusz, E.J., and Willaiams, M. 1991. [3H]CGP 39653: A new N‐methyl‐D‐aspartate antagonist radioligand with low nanomolar affinity in rat brain. Eur. J. Pharmacol. 192:19‐24.
   Watkins, J.C. and Evans, R.H. 1981. Excitatory amino acid transmitters. Annu. Rev. Pharmacol. Toxicol. 21:165‐204.
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