Benzodiazepine Binding to GABAA Receptors

Dietmar Benke1, Hanns Möhler1

1 University of Zurich, Zurich
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.16
DOI:  10.1002/0471141755.ph0116s35
Online Posting Date:  December, 2006
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Abstract

This unit describes ligand binding assays for the characterization of the benzodiazepine (BZ) site of GABAA receptors in the central nervous system. The Basic Protocol provides a procedure for analyzing benzodiazepine binding to GABAA receptors from brain. The experimental design of the method is suitable for all commercially available radioligands that interact with the BZ site of central GABAA receptors. By using the appropriate radioligand in combination with particular displacer compounds, individual (or groups of) GABAA receptor subtypes can be identified. Preparation of membrane suspensions for this procedure is described in a Support Protocol, and five Alternate Protocols are provided for the identification of diazepam‐sensitive GABAA receptors, the α1‐subtype of diazepam‐sensitive receptors, the α5 subtype of diazepam‐sensitive receptors, both diazepam‐sensitive and diazepam‐insensitive receptors, and diazepam‐insensitive receptors exclusively. Each protocol contains a brief description of the requirements for radioligand binding to these five types of BZ sites.

Keywords: benzodiazepine; BZ; GABAA; diazepam; radioligand binding; receptor subtype; LGIC

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Benzodiazepine Binding in Brain Membranes
  • Support Protocol 1: Preparation of Membranes
  • Alternate Protocol 1: Benzodiazepine Binding to Diazepam‐Sensitive GABAA Receptors
  • Alternate Protocol 2: Benzodiazepine Binding to Diazepam‐Sensitive GABAA Receptor Suptype Containing the α1 Subunit
  • Alternate Protocol 3: Benzodiazepine Binding to Diazepam‐Sensitive GABAA Receptor Subtypes Containing the α5 Subunit
  • Alternate Protocol 4: Benzodiazepine Binding to Diazepam‐Sensitive and Diazepam‐Insensitive GABAA Receptors
  • Alternate Protocol 5: Benzodiazepine Binding to All Diazepam‐Insensitive GABAA Receptors
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Benzodiazepine Binding in Brain Membranes

  Materials
  • Frozen membrane preparation (see protocol 2)
  • 50 mM Tris‐citrate buffer, pH 7.4 (see recipe), 4°C
  • Radioligands (see and Table 1.16.2)
  • Clonazepam (Sigma‐Aldrich) or Ro 15‐1788 (Sigma‐Aldrich) for determination of nonspecific binding
  • Unlabeled ligand (same compound as radioligand) for “cold” saturation binding assay
  • Tissue homogenizer (e.g., Polytron, Brinkmann)
  • Refrigerated centrifuge with rotor and tubes
  • 10 × 75–mm assay tubes
  • Whatman GF/B or GF/C glass fiber filters
  • Vacuum filtration device (e.g., Sartorius, Millipore) or cell harvester (e.g., Brandel, Scatron)
  • 6‐ml scintillation vials
  • Additional reagents and equipment for protein determination ( appendix 3A)

Support Protocol 1: Preparation of Membranes

  Materials
  • Fresh or frozen brain tissue
  • Tris‐citrate/sucrose buffer (see recipe), 4°C
  • 5 mM Tris‐citrate buffer, pH 7.4 (see recipe), containing 5 mM EDTA, 4°C
  • 50 mM Tris‐citrate buffer, pH 7.4 (see recipe), 4°C
  • Potter‐Elvehjem glass homogenizer with Teflon pestle and/or tissue homogenizer (e.g., Polytron, Brinkmann)
  • Refrigerated centrifuge with rotors and tubes
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Figures

Videos

Literature Cited

Literature Cited
   Benke, D., Fritschy, J.M., Trzeciak, A., Bannwarth, W., and Möhler, H. 1994. Distribution, prevalence and drug binding profile of GABAA receptor subtypes differing in β‐subunit variant. J. Biol. Chem. 269:27100‐27107.
   Benke, D., Honer, M., Michel, C., and Möhler, H. 1996. GABAA receptor subtypes differentiated by their γ‐subunit variants: Prevalence, pharmacology and subunit architecture. Neuropharmacology 35:1413‐1423.
   Benson, J.A., Low, K., Keist, R., Möhler, H., and Rudolph, U. 1998. Pharmacology of recombinant γ‐aminobutyric acidA receptors rendered diazepam‐insensitive by point‐mutated α‐subunits. FEBS Lett. 431:400‐404.
   Besnard, F., Avenet, P., Itier, V., Granger, P., Partiseti, M., Depoortere, H., Graham, D., and Langer, S.Z. 1996. GABAA receptor subtypes and the mechanism of action of zolpidem. In Zolpidem: An Update of Its Pharmacological Properties and Therapeutic Place in the Management of Insomnia (H. Freeman, A. J. Puech, and T. Roth, eds.) pp. 21‐31. Editions Scientifiques & Medicales, Elsevier, Paris.
   Fritschy, J.M. and Möhler, H. 1995. GABAA‐receptor heterogeneity in the adult rat brain: Differential regional and cellular distribution of seven major subunits. J. Comp. Neurol. 359:154‐194.
   Haefely, W., Kyburz, E., Gerecke, M., and Möhler, H. 1985. Recent advances in the molecular pharmacology of benzodiazepine receptors and in the structure‐activity relationships of their agonists and antagonists. Adv. Drug Res. 14:166‐322.
   McKernan, R.M. and Whiting, P.J. 1996. Which GABAA‐receptor subtypes really occur in the brain? Trends Neurosci. 19:139‐143.
   McPherson, G.A. 1986. Analysis of radioligand binding experiments: A collection of computer programs for the IBM PC. J. Pharmacol. Methods 14:213‐228.
   Möhler, H., Fritschy, J.M., Luscher, B., Rudolph, U., Benson, J., and Benke, D. 1996. The GABAA receptors from subunits to diverse functions. In Ion Channels, Vol. 4 (T. Narahashi, ed.) pp. 89‐113. Plenum, New York.
   Möhler, H., Fritschy, J.M., and Rudolph, U. 2002. A new benzodiazepine pharmacology. J. Pharmacol. Exp. Ther. 300:2‐8.
   Quirk, K., Blurton, P., Fletcher, S., Leeson, P., Tang, F., Mellilo, D., Ragan, C.I., and McKernan, R.M. 1996. [3H]L‐655,708, a novel ligand selective for the benzodiazepine site of GABAA receptors which contain the α5 subunit. Neuropharmacology 35:1331‐1335.
   Rudolph, U. and Möhler, H. 2006. GABA‐based therapeutic approaches: GABAA receptor subtype functions. Curr. Opin. Pharmacol. 6:18‐23.
   Skolnick, P., Hu, R.J., Cook, C.M., Hurt, S.D., Trometer, J.D., Lu, R.Y., Huang, Q., and Cook, J.M. 1997. [3H]RY 80: A high‐affinity, selective ligand for gamma‐aminobutyric acid(A) receptors containing α5 subunits. J. Pharmacol. Exp. Ther. 283:488‐493.
   Wieland, H.A., Luddens, H., and Seeburg, P.H. 1992. A single histidine in GABAA receptors is essential for benzodiazepine agonist binding. J. Biol. Chem. 267:1426‐1429.
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