Characterization of GABA Receptors

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

Described in this unit are ligand?binding assays for GABA_A , GABA_B , and the homomeric ? GABA_A (formerly GABA_C ) 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 GABA_A 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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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)
[³ H]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)
[³ H]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 CaCl₂
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)
[³ H]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 )

[³ H](–)‐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)
[³ H]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

Figure 1.7.1 Analysis of specific [³ H]muscimol binding to rat brain synaptic membranes (Beaumont et al., ). (A ) Saturation of specific [³ H]muscimol binding with increasing concentrations of [³ H]muscimol. Rat whole brain synaptic membrane suspensions (1.0 mg protein/tube) were incubated in Tris citrate (pH 7.1) containing various concentrations of [³ H]muscimol in the presence and absence of unlabeled GABA (200 µM). (B ) Scatchard plot of specific [³ H]muscimol binding from panel A. Dissociation constant ( K _d ) and maximum binding ( B _max ) values for high‐ and low‐affinity [³ H]muscimol binding sites were calculated using LIGAND.

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Figure 1.7.2 Analysis of specific sodium‐independent [³ H]GABA binding to rat brain synaptic membranes treated with 0.05% Triton X‐100 (Enna and Snyder, ). (A ) Saturation of specific [³ H]GABA binding with increasing concentrations of [³ H]GABA. (B ) Scatchard plot of specific [³ H]GABA binding from data show in panel A. Dissociation constant ( K _d ) and maximum binding ( B _max ) values for high‐ and low‐affinity [³ H]GABA binding sites were calculated using LIGAND.

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Figure 1.7.3 Analysis of specific [³ H]GABA binding to rat brain synaptic membranes (Bowery et al., ). (A ) Saturation of specific [³ H]GABA binding with increasing concentrations of [³ H]GABA. (B ) Scatchard plot of specific [³ H]GABA binding from panel A. Dissociation constant ( K _d ) and maximum binding ( B _max ) values for high‐ and low‐affinity [³ H]GABA binding sites were calculated using LIGAND.

View Image

Figure 1.7.4 Analysis of [³ H](–)‐baclofen binding to rat brain synaptic membranes (Bowery et al., ). (A ) Saturation of specific [³ H](–)‐baclofen binding with increasing concentrations of [³ H](–)‐baclofen. (B ) Scatchard plot of specific [³ H](–)‐baclofen binding from panel A. Dissociation constant ( K _d ) and maximum binding ( B _max ) values for high‐ and low‐affinity [³ H](–)‐baclofen binding sites were calculated using LIGAND.

View Image

Figure 1.7.5 Analysis of specific [³ H]GABA binding to rat cerebellar synaptic membranes in the presence of 40 µM isoguvacine (Drew and Johnston ). (A ) Saturation of specific [³ H]GABA binding with increasing concentrations of [³ H]GABA. (B ) Scatchard plot of specific [³ H]GABA binding from panel A. Dissociation constant ( K _d ) and maximum binding ( B _max ) values for high‐ and low‐affinity [³ H]GABA binding sites were calculated using LIGAND.

View Image

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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Characterization of GABA Receptors

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited