Saturation Assays of Radioligand Binding to Receptors and Their Allosteric Modulatory Sites

Anthony Basile1

1 National Institute of Diabetes, Digestive, and Kidney Diseases, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 7.6
DOI:  10.1002/0471142301.ns0706s01
Online Posting Date:  May, 2001
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Abstract

The protocols in this unit describe methods for measuring radioligand binding to sites on the GABA‐A receptor complex. This system was chosen because of the fundamental role of GABA‐A receptors in central nervous system function and the presence of multiple binding sites on one supramolecular complex. However, these basic techniques can be used to analyze ligand binding to a wide variety of intracellular and extracellular sites associated with ion channels, transporters, or G proteins with relatively simple modifications of the protocols (e.g., tissue preparation, buffers, assay termination techniques, incubation times, and temperatures). A saturation assay is presented to examine ligand binding to the benzodiazepine site on the GABA‐A receptor, while a second protocol uses a similar technique to examine the binding site for GABA in this complex.

     
 
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Table of Contents

  • Basic Protocol 1: Saturation Analysis of Ligand Binding to the Benzodiazepine Site
  • Basic Protocol 2: Saturation Analysis of Ligand Binding to the GABAA Receptor
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Saturation Analysis of Ligand Binding to the Benzodiazepine Site

  Materials
  • Rat
  • 320 mM sucrose (optional; see recipe), 0° to 4°C
  • 50 mM Tris citrate buffer, pH 7.4 (see recipe); 0° to 4°C
  • 10 mM flunitrazepam (see recipe)
  • 100 nM [3H]flumazenil (i.e., [3H]Ro15‐1788; see recipe)
  • Scintillation fluid (e.g., Cytoscint, ICN Biomedicals)
  • Dissection tools
  • Tissue homogenizer (e.g., Polytron; Brinkmann)
  • Sorvall RC‐5C+ centrifuge and SS‐34 rotor (Du Pont), or equivalent
  • 12 × 75–mm disposable borosilicate glass test tubes, or 1 ml × 96–well polystyrene microtiter plates
  • Glass fiber filter strips (grade 32; Schleicher & Schuell)
  • Filter manifold (M‐24R; Brandel)
  • Liquid scintillation spectrometer (e.g., Beckman LS 6500) and scintillation vials

Basic Protocol 2: Saturation Analysis of Ligand Binding to the GABAA Receptor

  Materials
  • Rat
  • 50 mM Tris citrate buffer (see recipe), 0° to 4°C
  • 10 mM γ‐aminobutyric acid (GABA; see recipe)
  • [3H]Muscimol (NEN; 30 Ci/mmol)
  • 100 µM unlabeled muscimol (see recipe)
  • 0.03% polyethylenimine (see recipe)
  • Dissection tools
  • Tissue homogenizer (e.g., Polytron; Brinkmann)
  • Sorvall RC‐5C+ centrifuge and SS‐34 rotor (Du Pont), or equivalent
  • 12 × 75–mm disposable borosilicate glass test tubes, or 1 ml × 96–well microtiter plates
  • Glass fiber filter strips (grade 32; Schleicher & Schuell)
  • Filter manifold (M‐24R; Brandel)
  • Liquid scintillation spectrometer (e.g., Beckman LS 6500) and scintillation vials
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Figures

Videos

Literature Cited

Literature Cited
   Basile, A.S. and Skolnick, P. 1986. Subcellular localization of “peripheral‐type” binding sites for benzodiazepines in rat brain. J. Neurochem. 46:305‐308.
   Basile, A.S. and Skolnick, P. 1987. Preservation of “peripheral” benzodiazepine receptors: Differential effects of freezing on [3H]Ro 5‐4864 and [3H]PK 11195 binding. J. Pharmacol. Methods 17:149‐156.
   Beaumont, K., Chilton, W.S., Yamamura, H.I., and Enna, S.J. 1978. Muscimol binding in rat brain: Association with synaptic GABA receptors. Brain Res. 148:153‐162.
   Bennett, J.P. and Yamamura, H.I. 1985. Neurotransmitter, hormone or drug receptor binding methods. In Neurotransmitter Receptor Binding (H.I. Yamamura, S.J. Enna, and M.J. Kuhar, eds.) pp. 61‐89. Raven Press, New York.
   Ehrlich, P. 1913. Chemotherapeutics: Scientific principles, methods and results. Lancet 2:445‐451.
   Hanley, M. 1985. Peptide binding assays. In Neurotransmitter Receptor Binding (H.I. Yamamura, S.J. Enna, and M.J. Kuhar, eds.) pp. 91‐101. Raven Press, New York.
   Harris, B.D., Wong, G., Moody, E.J., and Skolnick, P. 1995. Different subunit requirements of volatile and nonvolatile anesthetics at γ‐aminobutyric acid type A receptors. Mol. Pharmacol. 47:363‐367.
   Lee, C.M., Javitch, J.A., and Snyder, S.H. 1983. 3H–substance P binding to salivary gland membranes. Mol. Pharmacol. 23:563‐569.
   O'Brien, R.A. 1986. Receptor Binding in Drug Research. Marcel Dekker, New York.
   Pert, C.B., Bowie, D.L., Pert, A., Morrell, J.L., and Gross, E. 1977. Agonist‐antagonist properties of N‐allyl‐[D‐Ala]‐2‐Met‐enkephalin. Nature 269:73‐75.
   Rosenthal, H.E. 1967. A graphic method for the determination and presentation of binding parameters in complex systems. Anal. Biochem. 20:525‐532.
   Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660‐672.
   Weiland, G.A., Minneman, K.P., and Molinoff, P.B. 1979. Fundamental differences between the molecular interactions of agonists and antagonists with the β‐adrenergic receptor. Nature 281:114‐117.
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