Saturation Assays of Radioligand Binding to Receptors and Their Allosteric Modulatory Sites

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

The protocols in this unit describe methods for measuring radioligand binding to sites on the GABA?A receptor complex. This system was chosen because of the fundamental role of GABA?A receptors in central nervous system function and the presence of multiple binding sites on one supramolecular complex. However, these basic techniques can be used to analyze ligand binding to a wide variety of intracellular and extracellular sites associated with ion channels, transporters, or G proteins with relatively simple modifications of the protocols (e.g., tissue preparation, buffers, assay termination techniques, incubation times, and temperatures). A saturation assay is presented to examine ligand binding to the benzodiazepine site on the GABA?A receptor, while a second protocol uses a similar technique to examine the binding site for GABA in this complex.

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Basic Protocol 1: Saturation Analysis of Ligand Binding to the Benzodiazepine Site
Basic Protocol 2: Saturation Analysis of Ligand Binding to the GABAA Receptor
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Saturation Analysis of Ligand Binding to the Benzodiazepine Site

Materials

Rat
320 mM sucrose (optional; see recipe ), 0° to 4°C
50 mM Tris citrate buffer, pH 7.4 (see recipe ); 0° to 4°C
10 mM flunitrazepam (see recipe )
100 nM [³ H]flumazenil (i.e., [³ H]Ro15‐1788; see recipe )
Scintillation fluid (e.g., Cytoscint, ICN Biomedicals)

Dissection tools
Tissue homogenizer (e.g., Polytron; Brinkmann)
Sorvall RC‐5C+ centrifuge and SS‐34 rotor (Du Pont), or equivalent
12 × 75–mm disposable borosilicate glass test tubes, or 1 ml × 96–well polystyrene microtiter plates
Glass fiber filter strips (grade 32; Schleicher & Schuell)
Filter manifold (M‐24R; Brandel)
Liquid scintillation spectrometer (e.g., Beckman LS 6500) and scintillation vials

Basic Protocol 2: Saturation Analysis of Ligand Binding to the GABAA Receptor

Materials

Rat
50 mM Tris citrate buffer (see recipe ), 0° to 4°C
10 mM γ‐aminobutyric acid (GABA; see recipe )
[³ H]Muscimol (NEN; 30 Ci/mmol)
100 µM unlabeled muscimol (see recipe )
0.03% polyethylenimine (see recipe )

Dissection tools
Tissue homogenizer (e.g., Polytron; Brinkmann)
Sorvall RC‐5C+ centrifuge and SS‐34 rotor (Du Pont), or equivalent
12 × 75–mm disposable borosilicate glass test tubes, or 1 ml × 96–well microtiter plates
Glass fiber filter strips (grade 32; Schleicher & Schuell)
Filter manifold (M‐24R; Brandel)
Liquid scintillation spectrometer (e.g., Beckman LS 6500) and scintillation vials

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Figures

Figure 7.6.1 Schematic representation of the binding sites on the GABA_A receptor complex and some representative compounds that interact with them. The GABA_A receptor complex is a heteropentamer consisting of combinations of α, β, and γ subunits forming a channel that is permeable to chloride ions. The β subunit contains binding sites for: (1) barbiturates (e.g., the depressant pentobarbital and the excitatory barbiturate DMBB), (2) neurosteroids (e.g., the anesthetic alphaxalone and endogenous steroid 5α‐THDOC), and (3) the ion channel blockers picrotoxin and TBPS. The binding of ligands (e.g., the agonist muscimol and antagonist bicuculline) to the GABA receptor requires both α and β subunits. Similarly, both α and γ subunits are required for optimal binding of the agonist diazepam and the antagonist flumazenil to the benzodiazepine receptor, and of halothane to the gaseous anesthetic site (Harris et al., ).

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Figure 7.6.2 The two representative types of equilibrium binding assays: the competition assay (A ) and the saturation assay (B ). In the competition assay, increasing concentrations of unlabeled ligand compete with a fixed concentration of radioligand for the binding site, resulting in a sigmoidally shaped decrease in the amount of radioligand specifically bound. The competition assay determines the concentration of unlabeled ligand that inhibits 50% of the radioligand binding (IC₅₀ ; 1 nM), the efficacy of radioligand binding inhibition ( I _max ; 100%), and the presence of cooperativity (or multiple binding sites: the Hill coefficient, n _H ; 1.1). The saturation assay employs increasing concentrations of radioligand to bind to a fixed amount of receptor. The total and nonspecific radioligand bindings are determined directly, while the specific radioligand binding is derived by subtracting the nonspecific binding from the total. Fitting a rectangular hyperbola to the binding data by nonlinear regression analysis allows the K _d (6.5 nM) and B _max (209 fmol/assay) to be determined.

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Figure 7.6.3 [³ H]Flumazenil binding to benzodiazepine receptors from rat brain homogenates: Saturation isotherms (A ) and a Scatchard plot (B ). Panel A displays the total and specific binding, which can be modeled by rectangular hyperbolas. Nonspecific binding of [³ H]flumazenil (diamonds) increases linearly with increasing concentration, but is <12% of the total binding. Subtracting the nonspecific binding of [³ H]flumazenil from the total binding yields the specific binding. Fitting a rectangular hyperbola to the specific binding data by nonlinear regression analysis yields a K _d = 2.77 nM and B _max = 252 fmol/assay. When the specific binding data is linearized by plotting the amount of [³ H]flumazenil bound on the x axis versus the bound [³ H]flumazenil/free [³ H]flumazenil on the y axis (panel B), the K _d = 3.01 nM and B _max = 260 fmol/assay.

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Figure 7.6.4 [³ H]Muscimol binding to GABA receptors from rat brain homogenates: Saturation isotherms (A ) and a Scatchard plot (B ). Panel A displays the total (square) and specific (circle) binding, which can be modeled by rectangular hyperbolas. Specific [³ H]muscimol binding was determined by subtracting the nonspecific binding from the total binding. The nonspecific binding of [³ H]muscimol increases linearly with increasing concentration, to a maximum of 59% of the total binding. Fitting a rectangular hyperbola to the specific binding data by nonlinear regression analysis yields: K _d1 = 42 nM; K _d2 = 218 nM; B _max1 = 129 fmol/assay; and B _max2 = 408 fmol/assay. When the specific binding data is linearized by plotting the amount of [³ H]muscimol bound on the x axis versus the bound/free ratio on the y axis (panel B), a distinct departure from linearity is noted, consistent with the presence of multiple radioligand binding sites.

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Figure 7.6.5 Dependence of specific [³ H]flumazenil binding to the benzodiazepine receptor on the tissue concentration. Increasing volumes (5 to 400 µl) of rat brain homogenate containing 8 to 640 µg of protein were added to assay tubes containing 1 nM [³ H]flumazenil. The data were plotted as the log protein concentration (µg) versus [³ H]flumazenil bound (fmol/assay), to which a sigmoidal curve can be fitted. Initially, specific radioligand binding levels are low. They then rapidly increase in a linear fashion with increasing protein concentration, asymptotically reaching a maximal level. Tissue concentrations that fall within the linear range of specific radioligand binding should be used in the binding assays.

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Figure 7.6.6 Increase in specific radioligand binding with time. Radioligand binding (in this case, 5 nM [³ H](+)‐pentazocine binding to the sigma receptor in guinea‐pig brain) increases exponentially with time, becoming maximal after 60 min. Based on this data, equilibrium binding assays employing this radioligand should be incubated for >60 min.

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Literature Cited

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