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 GABAA , GABAB , and the homomeric ? 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
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
Materials
Basic Protocol 1: Measurement of GABAA Receptor Binding in Rat Brain Membranes Using [3H]Muscimol
Materials
Alternate Protocol 1: Measurement of GABAA Receptor Binding in Rat Brain Membranes Using [3H]GABA
Additional Materials (also see protocol 1 )
Basic Protocol 2: Measurement of GABAB Receptor Binding in Rat Brain Membranes Using [3H]GABA
Materials
Alternate Protocol 2: Measurement of GABAB Receptor Binding in Rat Brain Membranes Using [3H]Baclofen
Additional Materials (also see protocol 3 )
Basic Protocol 3: Measurement of Homomeric ρ Subunit GABAA (Formerly GABAC) Receptor Binding in Rat Brain Membranes Using [3H]GABA
Materials
Support Protocol 1: Preparation of Membranes
Materials
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Figures
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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. View Image -
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. View Image -
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
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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. |