Characterization of GABA Receptors

S.J. Enna1, Kenneth E. McCarson1

1 University of Kansas Medical Center, Kansas City, Kansas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.7
DOI:  10.1002/0471141755.ph0107s63
Online Posting Date:  December, 2013
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Abstract

Described in this unit are ligand‐binding assays for GABAA, GABAB, and the homomeric ρ GABAA (formerly GABAC) receptor recognition sites in brain tissue. Although GABA binding sites are present in peripheral organs, most research is directed toward examining these receptors in the CNS. These assays may also be used to determine the affinity of an unlabeled compound for the GABA binding sites. Excluded from the unit are ligand‐binding assays for other components of the GABAA receptor complex, such as the benzodiazepine or ion‐channel binding sites. Curr. Protoc. Pharmacol. 63:1.7.1‐1.7.20. © 2013 by John Wiley & Sons, Inc.

Keywords: gamma amino butyric acid; neurotransmitter; muscimol; baclofen; CNS; ligand binding

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of GABAA Receptor Binding in Rat Brain Membranes Using [3H]Muscimol
  • Alternate Protocol 1: Measurement of GABAA Receptor Binding in Rat Brain Membranes Using [3H]GABA
  • Basic Protocol 2: Measurement of GABAB Receptor Binding in Rat Brain Membranes Using [3H]GABA
  • Alternate Protocol 2: Measurement of GABAB Receptor Binding in Rat Brain Membranes Using [3H]Baclofen
  • Basic Protocol 3: Measurement of Homomeric ρ Subunit GABAA (Formerly GABAC) Receptor Binding in Rat Brain Membranes Using [3H]GABA
  • Support Protocol 1: Preparation of Membranes
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of GABAA Receptor Binding in Rat Brain Membranes Using [3H]Muscimol

  Materials
  • Frozen membrane preparation (see protocol 6Support Protocol)
  • 50 mM Tris citrate buffer (pH 7.1 at 4°C; adjust pH of 1 M Tris base with a concentrated solution of citric acid at 4°C, then dilute 1:20)
  • [3H]Muscimol (5 to 20 Ci/mmol; PerkinElmer NEN)
  • (–)‐Bicuculline methiodide (Sigma‐Aldrich, or ICN Biomedicals)
  • Muscimol (unlabeled)
  • γ‐Amino‐n‐butyric acid (GABA; Sigma‐Aldrich, or ICN Biomedicals)
  • Test compound: unlabeled competitor (optional)
  • Scintillation cocktail
  • 50‐ml polypropylene centrifuge tubes
  • Tissue homogenizer (e.g., Polytron, Brinkmann; Tissumizer, Tekmar)
  • Refrigerated centrifuge (Sorvall RC‐5 with SS‐34 and SM‐24 rotors, or equivalent)
  • 13 × 100–mm borosilicate glass Pyrex culture tubes
  • Brandel cell harvester
  • Whatman GF/B glass fiber filters
  • Liquid scintillation counter and vials
  • Analysis software for binding data: e.g., LIGAND (Munson and Rodbard, ) or Prism (GraphPad)
  • Additional reagents and equipment for protein assay ( appendix 3A)

Alternate Protocol 1: Measurement of GABAA Receptor Binding in Rat Brain Membranes Using [3H]GABA

  Additional Materials (also see protocol 1)
  • 10% Triton X‐100 in Tris citrate buffer (see protocol 1 for buffer)
  • [3H]GABA (PerkinElmer NEN)
  • Tissue solubilizer (e.g., BTS‐450, Beckman, or equivalent)
  • Scintillation cocktail compatible with organic solvents
  • 15‐ml polypropylene centrifuge tubes

Basic Protocol 2: Measurement of GABAB Receptor Binding in Rat Brain Membranes Using [3H]GABA

  Materials
  • Frozen membrane preparation (see protocol 6Support Protocol)
  • 50 mM Tris·Cl (pH 7.4 at 25°C; appendix 2A)/2.5 mM CaCl 2
  • Triton X‐100
  • 0.05 M Tris citrate buffer (pH 7.1 at 4°C; adjust pH of 1 M Tris base with a concentrated solution of citric acid at 4°C, then dilute 1:20)
  • Isoguvacine (Sigma‐Aldrich, or ICN Biomedicals)
  • [3H]γ‐Amino‐n‐butyric acid (GABA; 25 to 40 Ci/mmol; PerkinElmer NEN)
  • (±)‐Baclofen or GABA (unlabeled; Sigma‐Aldrich, or ICN Biomedicals)
  • Test compound: unlabeled competitor (optional)
  • Tissue solubilizer (e.g., BTS‐450, Beckman, or equivalent)
  • Scintillation cocktail compatible with organic solvents
  • 50‐ and 15‐ml polypropylene centrifuge tubes
  • Tissue homogenizer (e.g., Polytron, Brinkmann; Tissumizer, Tekmar)
  • Refrigerated centrifuge (Sorvall RC‐5 with SS‐34 or SM‐24 rotors, or equivalent)
  • 37° and 25°C water baths
  • Liquid scintillation counter and vials
  • Analysis software for binding data: e.g., LIGAND (Munson and Rodbard, ) or Prism (GraphPad)
  • Additional reagents and equipment for protein assay ( appendix 3A)

Alternate Protocol 2: Measurement of GABAB Receptor Binding in Rat Brain Membranes Using [3H]Baclofen

  Additional Materials (also see protocol 3)
  • [3H](–)‐Baclofen (30 to 50 Ci/mmol; PerkinElmer NEN)

Basic Protocol 3: Measurement of Homomeric ρ Subunit GABAA (Formerly GABAC) Receptor Binding in Rat Brain Membranes Using [3H]GABA

  Materials
  • Frozen cerebellar membrane preparation (see protocol 6Support Protocol)
  • 50 mM Tris·Cl (pH 7.4 at 20°C; appendix 2A)
  • Isoguvacine (Sigma‐Aldrich, or ICN Biomedicals)
  • [3H]γ‐Amino‐n‐butyric acid (GABA; 25 to 40 Ci/mmol; PerkinElmer NEN)
  • GABA (unlabeled; Sigma‐Aldrich or ICN Biomedicals)
  • Test compound: unlabeled competitor (optional)
  • Scintillation fluid compatible with organic solvents
  • 50‐ml polypropylene centrifuge tubes
  • 20°C shaking water bath
  • Tissue homogenizer (Polytron, Brinkmann; Tissumizer, Tekmar)
  • Refrigerated centrifuge (Sorvall RC‐5 with SS‐34 and SM‐24 rotors, or equivalent)
  • Liquid scintillation counter and vials

Support Protocol 1: Preparation of Membranes

  Materials
  • Fresh brain sample
  • 0.32 M sucrose, ice cold
  • Potter‐Elvehjem glass homogenizer with Teflon pestle
  • Refrigerated centrifuge (Sorvall RC‐5 with SS‐34 and SM‐24 rotors or equivalent)
  • Tissue homogenizer (Polytron, Brinkmann; Tissumizer, Tekmar)
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Figures

Videos

Literature Cited

  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.
  Bittiger, H., Reymann, N., Forestl, W., and Mickel, S.J. 1993. 3H‐CGP 54626: A potent antagonist radioligand for GABAB receptors. Pharmacol. Commun. 2:23.
  Bowery, N.G., Hill, D.R., and Hudson, A.L. 1985. [3H](–)‐Baclofen: An improved ligand for GABAB sites. Neuropharmacology. 24:207‐210.
  Cutting, G.R., Lu, L., O'Hara, B.F., Kasch, L.M., Montrose‐Rafizadeh, C., Donovan, D.M., Shimada, S., Antonarakis, S.E., Guggino, W.B., Uhl, G.R., and Kazazian, H.H. 1991. Cloning of the γ‐aminobutyric acid (GABA) ρ 1 cDNA: A GABA receptor subunit highly expressed in the retina. Proc. Natl. Acad. Sci. U.S.A. 88:2673‐2677.
  Drew, C.A. and Johnston, G.A.R. 1992. Bicuculline‐ and baclofen‐insensitive γ‐aminobutyric acid binding to rat cerebellar membranes. J. Neurochem. 58:1087‐1092.
  Enna, S.J. and Snyder, S.H. 1975. Properties of γ‐aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res. 100:81‐97.
  Enna, S.J. and Snyder, S.H. 1977. Influences of ion, enzymes and detergents on γ‐aminobutyric acid receptor binding in synaptic membranes of rat brain. Mol. Pharmacol. 13:442‐453.
  Falch, E. and Krogsgaard‐Larsen, P. 1982. The binding of the GABA agonist [3H]THIP to rat brain synaptic membranes. J. Neurochem. 38:1123‐1129.
  Kaupmann, K., Huggel, K., Heid, J., Flor, P.J., Bischoff, S., Mickel, S.J., McMaster, G., Angst, C., Bittiger, H., Froestl, W., and Bettler, B. 1997. Expression cloning of GABAB receptors uncovers similarity to metabotropic glutamate receptors. Nature 386:239‐246.
  Krogsgaard‐Larsen, P., Snowman, A., Lummis, S.C., and Olsen, R.W. 1981. Characterization of the binding of the GABA agonist [3H]piperidine‐4‐sulfonic acid (P4S) to bovine brain synaptic membranes. J. Neurochem. 37:401‐409.
  Krogsgaard‐Larsen, P., Jacobsen, P., and Falch, E. 1983. Structure‐activity requirements of the GABA receptor. In The GABA Receptors (S.J. Enna, ed.) pp. 149‐176. Humana Press, Totowa, N.J.
  Möhler, H. and Okada, T. 1977. Properties of γ‐aminobutyric acid receptor binding with (+)‐[3H]bicuculline methiodide in rat cerebellum. Mol. Pharmacol. 14:256‐265.
  Möhler, H., Battersby, M.K., and Richards, J.G. 1980. Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label. Proc. Natl. Acad. Sci. U.S.A. 77:1661‐1670.
  Möhler, H., Benke, D., Benson, J., Lüscher, B., Rudolph, U., and Fritschy, J.M. 1997. Diversity in structure, pharmacology, and regulation of GABAA receptors. In The GABA Receptors, 2nd ed. (S.J. Enna and N.G. Bowery, eds.) pp. 11‐36. Humana Press, Totowa, N.J.
  Möhler, H., Fritschy, J.‐M., Crestani, F., Hensch, T., and Rudolph, U. 2004. Specific GABAA circuits in brain development and therapy. Biochem. Pharmacol. 68:1685‐1690.
  Munson, P.J. and Rodbard, D. 1980. LIGAND: A versatile computerized approach for characterization of ligand‐binding systems. Anal. Biochem. 107:220‐239.
  Olsen, R.W. 1981. The GABA postsynaptic membrane receptor‐ionophore complex: Site of action of convulsant and anticonvulsant drugs. Mol. Cell. Biochem. 39:261‐279.
  Polenzani, L., Woodward, R.M., and Miledi, R. 1991. Expression of mammalian γ‐aminobutyric acid receptors with distinct pharmacology in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 88:4318‐4322.
  Tan, K., Rudolph, U., and Luscher, C. 2011. Hooked on benzodiazepines: GABAA receptor subtypes and addiction. Trends Pharmacol. Sci. 34:188‐197.
  Ticku, M.K., Ban, M., and Olsen, R.W. 1978. Binding of [3H] α‐dihydropicrotoxinin, a γ‐aminobutyric acid synaptic antagonist, to rat brain membranes. Mol. Pharmacol. 14:391‐402.
Key References
  Enna and Snyder, 1975. See above.
  Provides detailed description and appropriate citations for preparation of crude P2 membrane preparation from rat brain tissue.
  Enna and Snyder, 1977. See above.
  Details the effect of detergents on GABA receptor binding.
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