Benzodiazepine Binding to GABAA Receptors
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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 ?1?subtype of diazepam?sensitive receptors, the ?5 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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PDF or HTML at Wiley Online LibraryTable 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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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Benzodiazepine Binding in Brain Membranes
Materials
Support Protocol 1: Preparation of Membranes
Materials
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Figure 1.16.1 Classification by distinctive affinities of benzodiazepine site ligands of native GABAA receptor subtypes. The subunit combinations representing the respective receptor subtypes are indicated. αx or βx: subunit isoform has not been defined, can be any α(1‐6) or β(1‐3) subunit. View Image -
Figure 1.16.2 Analysis of specific [3 H]flumazenil binding to mouse brain membranes. (A ) Analysis of [3 H]flumazenil saturation binding. Membranes prepared from whole mouse brains (∼0.1 mg protein/tube) were incubated in 50 mM Tris‐citrate, pH 7.4, containing various concentrations of [3 H]flumazenil in the presence and absence of 10 µM clonazepam to determine total and nonspecific binding. (B ) Scatchard plot of specific [3 H]flumazenil binding shown in panel A. The dissociation constant ( K D ) and maximum number of binding sites ( B max ) using these diazepam‐sensitive GABAA receptors were determined using the computer program LIGAND. View Image -
Figure 1.16.3 Scatchard plot analysis of [3 H]Ro 15‐4513 saturation binding data to mouse brain membranes. (A ) Scatchard plot of saturation binding of [3 H]Ro 15‐4513 to diazepam‐sensitive and diazepam‐insensitive GABAA receptors. Membranes prepared from whole mouse brains (∼0.1 mg protein/tube) were incubated in 50 mM Tris‐citrate, pH 7.4, containing various concentrations of [3 H]Ro 15‐4513 in the presence and absence of 10 µM Ro 15‐1788 to determine total and nonspecific binding. (B ) Scatchard plot of saturation binding of [3 H]Ro 15‐4513 to diazepam‐insensitive GABAA receptors. Membranes prepared from whole mouse brains (∼0.1 mg protein/tube) were incubated in 50 mM Tris‐citrate, pH 7.4, containing various concentrations of [3 H]Ro 15‐4513 in the presence of 10 µM 10 diazepam to block all diazepam‐sensitive receptors. Nonspecific [3 H]Ro 15‐4513 binding was determined in the presence of 10 µM Ro 15‐1788. The dissociation constant ( K D ) and maximum number of binding sites ( B max ) were determined using the computer program LIGAND. View Image -
Figure 1.16.4 Scatchard plot analysis of [3 H]L655,708 binding to mouse brain membranes. [3 H]L655,708 binds with high affinity ( K D = 0.5 nM) selectively to GABAA receptors containing the α5 subunit. However, at high concentrations, [3 H]L655,708 also interacts with receptors containing the α1, α2, and α3 subunit (low affinity component of the curve, K D = 58 nM) View Image
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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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