Characterization of the Picrotoxin Site of GABAA Receptors
UNIT 1.18
Ashok K. Mehta1 and Maharaj K. Ticku1 1
The University of Texas Health Science Center, San Antonio, Texas
ABSTRACT This unit describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd , Bmax determination), association kinetics, dissociation kinetics, and competition assays (IC50 , Ki determination) are included. Curr. Protoc. C 2013 by John Wiley & Sons, Inc. Pharmacol. 63:1.18.1-1.18.18. Keywords: GABAA receptor r picrotoxin binding site r allosteric regulation r convulsants
INTRODUCTION NOTE: This unit was updated by the editors of Current Protocols in Pharmacology in 2013 to document changes related to the subject matter since the original publication and also to reflect current “state of the art” reference compounds, suppliers, and literature citations. This unit describes an in vitro assay for characterization of the picrotoxin site of γ-aminobutyric acid A (GABAA ) receptors in rat brain membranes using various radioligands, such as [35 S]t-butylbicyclophosphorothionate (35 STBPS or [35 S]TBPT), [3 H]t-butylbicycloorthobenzoate([3 H]TBOB), 4 -ethynyl-4-n-[2,3-3 H2 ]propylbicycloorthobenzoate ([3H]EBOB), and [3 H]3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3 H]BIDN). GABAA receptors are transmembrane hetero-oligomeric pentameric subunit assemblies derived from various subunits (such as α1 to α6 , β1 to β3 , γ1 to γ3 , δ, ɛ, π, and ρ1 to ρ3 ), and the β-subunit of GABAA receptors has been suggested as a necessary requirement for the picrotoxin site (Slany et al., 1995; Zezula et al., 1996). The four Basic Protocols describe procedures for investigating the picrotoxin site of GABAA receptors and its interaction with various compounds using [35 S]TBPS as a radioligand. Three Alternate Protocols describe the use of [3 H]TBOB (Alternate Protocol 1), [3 H]EBOB (Alternate Protocol 2), and [3 H]BIDN (Alternate Protocol 3). Preparation of the membrane suspension for this assay is described in a Support Protocol.
Update There has been a resurgence of interest in the GABAA receptor picrotoxin binding site with the growing interest in discovering and developing allosteric, as opposed to orthosteric, GABA receptor agonists and antagonists. The protocols detailed in this unit describing binding assays for characterizing the picrotoxin binding site remain current. The ligand of choice for labeling this site is still [35 S]TBPS, although [3 H]TBOB and [3 H]EBOB are occasionally employed as well. As with most binding assays, all of these protocols can be conducted using multi-well microplates instead of a cell harvester. CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer. Current Protocols in Pharmacology 1.18.1-1.18.18, December 2013 Published online December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471141755.ph0118s63 C 2013 John Wiley & Sons, Inc. Copyright
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BASIC PROTOCOL 1
SATURATION BINDING OF [35 S]TBPS TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES This protocol describes an in vitro assay for labeling the picrotoxin site of GABAA receptors in rat brain membranes using [35 S]TBPS as the radioligand. It has also been shown that this ligand can be employed to label GABAA receptors in post-mortem brain tissue, although the binding site in human tissue is more labile to degradation than that of rat (Atack et al., 2007). Picrotoxinin is used to define nonspecific binding. This protocol can be utilized to determine the Kd (affinity) of radioligands in saturation binding experiments (see UNIT 1.3). It is advisable to use approximately ten concentrations of radioligand ranging from 10-fold below to 20-fold above the estimated Kd value. The final volume of the binding assays for routine experiments is 1 ml, but it can be scaled to 0.5 ml or 2.0 ml, depending on the cost and desired accuracy. Pharmaceutical companies even use 100- or 200-µl assays for routine high-throughput screening.
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl, pH 7.4 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN Life) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester) Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer data graphing/fitting program such as LIGAND; Munson and Rodbard, 1980, or Prism (GraphPad) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: Picrotoxinin should first be dissolved to 100 mM in dimethylsulfoxide (DMSO), but the final concentration of DMSO should not exceed 0.1% (v/v) in the 1-ml assay.
Prepare membrane suspension for binding assay 1. On the day of the assay, thaw a frozen membrane preparation and resuspend in 20 to 50 vol ice-cold 50 mM Tris·Cl in a 25-ml centrifuge tube using a tissue homogenizer (midpoint setting). 2. Centrifuge homogenate 30 min at 140,000 × g, 4°C. 3. Discard supernatant, suspend resultant pellet in ice-cold 50 mM Tris·Cl, and centrifuge 30 min at 140,000 × g, 4°C. Repeat this step one more time. Multiple washings of the tissue are necessary to remove endogenous GABA that is present in high concentrations in brain tissue; otherwise, residual GABA in the membrane preparation will interfere with the binding assay. However, it is adequate to wash the membrane suspension two times on the day of assay (omitting the third wash) for preliminary results/screening, provided the membrane is prepared over a 2-day period using the freeze-thaw cycle described (see Support Protocol). Picrotoxin Site of GABAA Receptors
4. Resuspend the final pellet in ice-cold 50 mM Tris·Cl to yield a protein concentration of 2 to 3 mg/ml.
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One gram of tissue yields 20 mg protein. Protein concentration may be measured using BCA (Pierce), Lowry, Bradford, or other suitable assay with BSA as reference standard (or γ -globulin in the case of the Bradford assay). See APPENDIX 3A for protein assays. The final protein content in the 1.0-ml assay mixture should be in the range of 0.2 to 0.3 mg.
Generate binding-site saturation data 5. To measure total binding: Prepare triplicate 12 × 75–mm borosilicate glass culture tubes for total binding by combining the following reagents, using ten concentrations of radioligand in the range indicated. 100 µl 1.5 M KCl (150 mM final) 0.4 to 100 µl 1.2 µM [35 S]TBPS (0.5 to 120 nM final) 50 mM Tris·Cl to 0.9 ml. Saturation data are obtained using increasing concentrations of the radioligand in the absence (total binding) and presence (blank; see step 6) of a saturating concentration of displacer (100 µM picrotoxinin). Total and nonspecific binding are determined in parallel for each concentration of radioligand. It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent. In the case of [35 S]TBPS (Kd 20 nM), a concentration range of 0.5 to 120 nM yields reliable and reproducible saturation kinetic data. In general, ten concentrations of a radioligand covering a range from 10-fold below to 20-fold above its Kd value are usually sufficient for saturation binding assays. To investigate the saturation binding kinetics of a test compound, replace [35 S]TBPS with a radiolabeled test compound using concentrations in this range.
6. To measure nonspecific binding: Prepare parallel triplicate tubes for nonspecific binding at the same radioligand concentrations. Combine reagents as described in step 5, but add 100 µl of 1 mM picrotoxinin (final 100 µM) and decrease the Tris·Cl to maintain a 0.9-ml volume. 7. To initiate the binding assay, add 100 µl membrane suspension (0.2 to 0.3 mg protein) to each assay tube (final 1.0 ml), gently vortex to mix the contents, and incubate 180 min at room temperature (24°C) to achieve binding equilibrium. [35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
8. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper with polyethyleneimine (PEI).
9. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
10. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 11. Calculate the specific radioligand binding (dpm) at each radioligand concentration by subtracting nonspecific binding from total binding. Receptor Binding
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Figure 1.18.1 [35 S]TBPS binding to rat brain membranes. (A) Saturation of specific [35 S]TBPS binding with increasing concentrations of [35 S]TBPS in the absence and presence of picrotoxinin (100 µM). (B) Scatchard plot of specific [35 S]TBPS binding from panel A. Kd and Bmax values were found to be 25 nM and 1.5 pmol/mg protein, respectively. (C) Concentration-dependent inhibition of specific [35 S]TBPS binding by picrotoxinin and stereoisomers of etomidate in rat brain membranes. IC50 values of picrotoxinin, (+)etomidate, and (–)etomidate were found to be 0.4 µM, 9 µM, and 100 µM, respectively. Reprinted from Ramanjaneyulu and Ticku (1984a) with permission from Raven Press.
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12. Convert specific binding to the concentration of radioligand bound. Use a Scatchard transformation plot (bound ligand versus bound/free ligand) to determine the affinity constant of the radioligand (Kd ) and the maximum number of binding sites (Bmax ) by using a suitable iterative fitting computer program, such as LIGAND
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(Munson and Rodbard, 1980) or Prism (GraphPad) or linear regression analysis (see Fig. 1.18.1A,B). See UNIT 1.3 for a thorough explanation of the calculations. For a detailed procedure to analyze the data using computer programs, the experimenter is advised to refer to the program manuals. Kd values can also be derived from the association and dissociation kinetics data as described below (see Basic Protocols 3 and 4).
COMPETITION ASSAYS FOR [35 S]TBPS BINDING TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES
BASIC PROTOCOL 2
This protocol can be utilized to determine the potency (IC50 or Ki ) of unlabeled test compounds in competition experiments. This procedure requires an unlabeled test compound or reference compound, a radioligand (e.g., 4 nM [35 S]TBPS for labeling picrotoxin sites), and a reference displacer (e.g., 100 µM picrotoxinin) to define nonspecific binding by using a concentration sufficient to completely displace the radioligand from the receptors. For determining the IC50 or Ki value of a test compound, ten concentrations of the unlabeled compound, covering a range from 10-fold below to 20-fold above its estimated IC50 value, are usually sufficient to determine reliable and reproducible values. Furthermore, the radioligand concentration should be less than the Kd value of the radioisotope (approximately one-tenth to one-half of the Kd value, which is 20 nM for [35 S]TBPS).
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl, pH 7.4 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN) in 50 mM Tris·Cl, pH 7.4 Unlabeled competitor (test compound) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester) Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer program (e.g., DeltaGraph; Delta Point) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: If unlabeled competitor is insoluble in 50 mM Tris·Cl, dimethylsulfoxide (DMSO) can be added. Picrotoxinin should first be dissolved to 100 mM in DMSO, but in all cases, the final concentration of DMSO should not exceed 0.1% (v/v) in the final 1-ml assay volume.
Prepare membrane suspension for binding assay 1. Prepare membranes as described (see Basic Protocol 1, steps 1 to 4). Generate competitive binding data 2. To measure total binding: Prepare a set of triplicate 12 × 75–mm borosilicate glass culture tubes for total binding in the absence (zero concentration of competitor)
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and increasing concentrations of unlabeled competitor by combining the following reagents:
100 µl 1.5 M KCl (150 mM final) 3.3 µl 1.2 µM [35 S]TBPS (4 nM final) Various concentrations of unlabeled competitor, including zero 50 mM Tris·Cl to 0.9 ml. Use ten concentrations of unlabeled competitor in a range from 10-fold below to 20-fold above its estimated IC50 or Ki . It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent.
3. To measure nonspecific binding: Prepare a set of parallel triplicate tubes for nonspecific binding. Combine reagents as described in step 2, but replace the unlabeled competitor with 100 µl of 1 mM picrotoxinin (final 100 µM). 4. To initiate the binding assay, add 100 µl membrane suspension (0.2 to 0.3 mg protein) to each assay tube (final 1.0 ml), gently vortex to mix the contents, and incubate 180 min at room temperature (24°C) to achieve binding equilibrium. [35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
5. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper with polyethyleneimine (PEI).
6. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
7. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 8. Calculate the specific radioligand binding (dpm) at each concentration of competitor by subtracting nonspecific binding from total binding. 9. Calculate the percentage inhibition of the specific radioligand binding by each concentration of competitor. Plot the percentage inhibition versus competitor concentration, and calculate the concentration that inhibits 50% of the specific radioligand binding (IC50 ) using a suitable computer program such as DeltaGraph. Calculate the inhibitory constant (Ki ) using the equation: Ki = IC50 /(1 + [L]/Kd ), where [L] is the molar concentration of radioligand and Kd is the affinity constant. See Figure 1.18.1C for a representative plot. Caution should be exercised in setting the limits for various variables while analyzing the data using such computer programs, because varied results may be obtained if unrealistic values are set. BASIC PROTOCOL 3 Picrotoxin Site of GABAA Receptors
ASSOCIATION KINETICS OF BINDING TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES This protocol describes an in vitro assay for determining the association kinetics of binding (association rate constant and affinity constant) of various radioligands to the
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picrotoxin site of GABAA receptors in rat brain membranes. These studies are useful in investigating the pharmacological interaction of GABAergic drugs with the picrotoxin site.
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl, pH 7.4 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN) in 50 mM Tris·Cl, pH 7.4 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester) Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer program (e.g., DeltaGraph; Delta Point) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: Picrotoxinin should first be dissolved to 100 mM in dimethylsulfoxide (DMSO), but the final concentration of DMSO should not exceed 0.1% (v/v) in the 1-ml assay.
Prepare membrane suspension for binding assay 1. Prepare membranes as described (see Basic Protocol 1, steps 1 to 4). Generate association kinetics data 2. To measure total binding: Prepare triplicate 12 × 75–mm borosilicate glass culture tubes for total binding for each time interval by combining the following reagents: 100 µl 1.5 M KCl (150 mM final) 3.3 µl 1.2 µM [35 S]TBPS (4 nM final) 50 mM Tris·Cl to 0.9 ml. For association kinetics, data are obtained in the absence (total binding) and presence (nonspecific binding) of a saturating concentration of displacer (100 µM picrotoxinin) using multiple incubation times. Total and nonspecific binding are determined in parallel at each time interval using a single concentration of [35 S]TBPS (4 nM final). It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent. To investigate the association kinetics of a test compound, replace [35 S]TBPS with a radiolabeled test compound. The optimum concentration should be determined using Basic Protocol 1.
3. To measure nonspecific binding: Prepare parallel triplicate tubes for nonspecific binding for each time interval. Combine reagents as described in step 2, but add 100 µl of 1 mM picrotoxinin (final 100 µM) and decrease the Tris·Cl to maintain a 0.9-ml volume. 4. To initiate the binding assay, add 100 µl membrane suspension (0.2 to 0.3 mg protein) to each assay tube (final 1.0 ml), gently vortex to mix the contents, and incubate corresponding nonspecific and total binding tubes for different periods of time (e.g., 10, 20, 40, 60, 90, 120, and 180 min) at room temperature. Current Protocols in Pharmacology
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Time (min) Figure 1.18.2 Analysis of association and dissociation kinetics of [35 S]TBPS binding to rat cortical membranes. (A) Association of [35 S]TBPS (4 nM) to rat cortical membranes at 25°C. The apparent association rate constant (kapp ) and association rate constant (k1 ) of [35 S]TBPS were found to be 0.0183 min–1 and 3.6 × 106 min–1 M–1 , respectively. (B) Dissociation of [35 S]TBPS (3 nM) binding from rat cortical membranes by pentylenetetrazole (PTZ; 6 mM), GABA (10 µM), and etazolate (100 µM) at 25°C. The respective half-lives of dissociation with these drugs were found to be 68 min, 1.3 min (first phase)/12 min (second phase), and 0.7 min (first phase)/20 min (second phase). Reprinted from Maksay and Ticku (1985) with permission from Raven Press.
[35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
Picrotoxin Site of GABAA Receptors
Execute the timing of the assay assembly in such a way that the termination of binding reaction in the assay tubes can be completed in a short time without any significant interruption in the filtration process. It usually takes 2 to 3 min to process one rack of 48 assay tubes using a vacuum filtration device with 48 slots (e.g., Brandel cell harvester model MB-48L). Therefore, a gap of 4 to 5 min should be maintained between different racks of assay tubes. The time course must include a 180-min time point so that Be (step 9) can be calculated.
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5. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper with polyethyleneimine (PEI).
6. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
7. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 8. Calculate the specific radioligand binding (dpm) by subtracting nonspecific binding from total binding. 9. Plot ln[(Be – Bt )/Be ] against time t, where Be and Bt are specific binding at equilibrium and at time t, respectively (see Fig. 1.18.2A). Calculate the apparent association rate constant (kapp ) from the slope of the resulting line. 10. Determine the association rate constant (k1 ) using the equation kapp = k1 [L] + k–1 , where [L] is the molar concentration of radioligand and k–1 is the dissociation rate constant (see Basic Protocol 4). Any suitable computer program such as DeltaGraph can be used for analyzing the association kinetic data. However, caution should be exercised in setting the limits for various variables while analyzing the data using such computer programs because varied results may be obtained if unrealistic values are set.
DISSOCIATION KINETICS OF BINDING TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES
BASIC PROTOCOL 4
This protocol describes an in vitro assay for determining the dissociation kinetics of binding (dissociation rate constant and affinity constant) of various radioligands to the picrotoxin site of GABAA receptors in rat brain membranes. These studies are utilized to investigate whether GABAergic drugs bind to the picrotoxin site or modulate this site as a result of their binding to a distinct, but allosterically coupled, site on the GABAA receptor.
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN) in 50 mM Tris·Cl 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Unlabeled test compound in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester)
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Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer program (e.g., DeltaGraph; Delta Point) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: If unlabeled competitor is insoluble in 50 mM Tris·Cl, dimethylsulfoxide (DMSO) can be added. Picrotoxinin should first be dissolved to 100 mM in DMSO, but in all cases, the final concentration should not exceed 0.1% (v/v) in the final 1-ml assay volume.
Prepare membrane suspension for binding assay 1. Prepare membranes as described (see Basic Protocol 1, steps 1 to 4). Generate dissociation kinetics data 2. To measure total binding: Prepare and label triplicate 12 × 75–mm borosilicate glass culture tubes for total binding and add the following reagents: 100 µl 1.5 M KCl (150 mM final) 3.3 µl 1.2 µM [35 S]TBPS (4 nM final) 50 mM Tris·Cl to 0.9 ml. For dissociation kinetics, equilibrium data are obtained in the absence (total binding) and presence (nonspecific binding) of a saturating concentration of displacer (100 µM picrotoxinin), and binding data are obtained for various time points in the presence of a saturating concentration of an unlabeled test compound (displacer) that causes dissociation of the bound radioligand. Total and nonspecific binding are determined in parallel using a single concentration of [35 S]TBPS (4 nM final). The total assay volume will be 1 ml after the addition of membrane suspension (step 5). It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent. To investigate the dissociation kinetics of a test compound, replace [35 S]TBPS with a radiolabeled test compound. The optimum concentration should be determined using Basic Protocol 1.
3. To measure nonspecific binding: Prepare triplicate tubes for nonspecific binding. Combine reagents as described in step 2, but add 100 µl of 1 mM picrotoxinin (final 100 µM) and decrease the Tris·Cl to maintain the appropriate volume. 4. To measure dissociation kinetics: For each time interval (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 60, 80, and 100 min), prepare triplicate tubes as in step 2, but decrease Tris·Cl to allow for addition of unlabeled test compound (step 6). 5. Initiate the binding assay in all tubes by adding 100 µl membrane suspension (0.2 to 0.3 mg protein). Gently vortex to mix the contents, and begin to incubate at room temperature. [35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
6. Initiate the dissociation of [35 S]TBPS binding at the appropriate times by adding a saturating concentration of unlabeled test compound (displacer) to the tubes prepared in step 4. Vortex gently. Continue to incubate all tubes for a total of 180 min.
Picrotoxin Site of GABAA Receptors
Time the addition of displacer such that the membrane suspension is incubated for 180 min with [35 S]TBPS and for a specified time with a displacer, and so that all the tubes, including those for total and nonspecific binding, are ready for the termination
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of the binding reaction without any significant interruption in the filtration process. For example, in order to obtain the data point representing the dissociation of the [35 S]TBPS binding in 100 min, add membrane suspension to the tubes containing buffer, KCl, and [35 S]TBPS (with and without picrotoxinin). Vortex the tubes gently. Wait for 80 min, and then add displacer. Vortex the tubes gently, incubate an additional 100 min, and then terminate the binding reaction.
7. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize additional dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper in polyethyleneimine (PEI).
8. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
9. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 10. Calculate the specific radioligand binding (dpm) by subtracting nonspecific binding from total binding (Be ) and from each time interval (Bt ). 11. Plot ln[Bt /Be ] versus time t, where Bt and Be are the amount of specific binding at time t and at equilibrium, respectively. Calculate the rate constant of dissociation (k–1 ) from the slope of the resulting line (see Fig. 1.18.2B). Any suitable computer program such as DeltaGraph can be used for analyzing the dissociation kinetic data. However, caution should be exercised in setting the limits for various variables while analyzing the data using such computer programs because varied results may be obtained if unrealistic values are set.
12. Calculate the affinity constant (Kd ) of the radioligand by dividing the dissociation rate constant (k–1 ) by the association rate constant (k1 ).
CHARACTERIZATION OF THE PICROTOXIN SITE OF GABAA RECEPTORS USING [3 H]TBOB
ALTERNATE PROTOCOL 1
[3 H]t-Butylbicycloorthobenzoate ([3 H]TBOB) and [35 S]TBPS label the same picrotoxin site on the GABAA receptor (Lawrence et al., 1985). However, [3 H]TBOB has a much longer radioactive half-life, and binding assays conducted at 0°C, 24°C, and 37°C yield similar Kd values, whereas [35 S]TBPS has negligible binding at 0°C. Furthermore, a shorter incubation time is needed for [3 H]TBOB binding (90 min versus 180 min at 24°C). In spite of these advantages, [35 S]TBPS is more commonly used as a research tool due to its relatively lower Kd value (20 nM versus 60 nM) at 24°C, and because [3 H]TBOB binding is considerably less sensitive than [35 S]TBPS binding to inhibition by GABA, muscimol, and Ro 5-4864, due to differences in the interactions of these radioligands with the identical picrotoxin site of the GABAA receptor (Squires et al., 1983; Ticku and Ramanjaneyulu, 1984; Lawrence et al., 1985). However, [3 H]TBOB is a superior radioligand for autoradiographic studies, due to better resolution. [3 H]TBOB is available commercially (20 to 60 Ci/mmol; GE Healthcare Life Sciences; PE NEN Life Sciences). For [3 H]TBOB binding assays, follow Basic Protocols 1 to 4, replacing [35 S]TBPS with [3 H]TBOB. The assay buffer (50 mM Tris·Cl, pH 7.4) should be adjusted to contain
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0.5 M NaCl at all stages. Incubation should be carried out for either 90 min at 24°C, 120 min at 0°C, or 60 min at 37°C. Nonspecific binding can be carried out using picrotoxinin (100 µM) or unlabeled TBOB (4 µM). Dimethylsulfoxide (DMSO) is used to dissolve picrotoxinin as well as TBOB, with a final concentration of DMSO not exceeding 0.1% (v/v) in the final 1-ml assay volume. ALTERNATE PROTOCOL 2
CHARACTERIZATION OF THE PICROTOXIN SITE OF GABAA RECEPTORS USING [3 H]EBOB 4 -Ethynyl-4-n-[2,3-3 H2 ]propylbicycloorthobenzoate ([3 H]EBOB), also called [3 H]ethynylbicycloorthobenzoate, is structurally related to TBOB. It has high affinity (Kd 2 nM) for the picrotoxin site on the GABAA receptor and is available commercially (30 to 60 Ci/mmol; Perkin Elmer NEN). Besides having a longer radioactive half-life and higher affinity than [35 S]TBPS, [3 H]EBOB binds to both vertebrate and invertebrate GABAA receptors (Yagle et al., 2003). For [3 H]EBOB binding assays, follow Basic Protocols 1 to 4, replacing [35 S]TBPS by [3 H]EBOB. The assay buffer (50 mM Tris·Cl, pH 7.4) should be adjusted to contain 0.3 M NaCl at all stages. Incubation should be carried out for 120 min at 24°C or 90 min at 37°C. Nonspecific binding can be carried out using picrotoxinin (100 µM), unlabeled EBOB (2 µM), or lindane (5 µM; Sigma). If DMSO is used to dissolve compounds, its final concentration should not exceed 0.1% (v/v) in the final 1-ml assay volume.
ALTERNATE PROTOCOL 3
CHARACTERIZATION OF PICROTOXIN SITE OF GABAA RECEPTOR USING [3 H]BIDN [3 H]3,3-bis-Trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3 H]BIDN) is suitable for studies of GABA-gated chloride channels of vertebrates and insects. The cage convulsants (TBPS, and TBOB) and picrotoxinin displace [3 H]BIDN binding in rat membranes, but not in insect membranes, suggesting that the convulsant site of the GABAA receptor is somewhat different in insects as compared to rats. However, dieldrin displaces [3 H]BIDN binding competitively in insect membranes. Unlike picrotoxin, which antagonizes a variety of ligand-gated chloride channels such as vertebrate glycine-gated and invertebrate L-glutamate-gated chloride channels, BIDN blocks only GABA-gated chloride channels in vertebrates and insects (Rauh et al., 1997). Thus, [3 H]BIDN offers the advantage of being selective for GABA-gated chloride channels, and can be utilized to investigate the pharmacology of chloride channels in vertebrates as well as in insects (Rauh et al., 1997; Hamon et al., 1998). However, its low affinity (higher Kd value) for rat brain membranes limits its use as an experimental tool. Kd values for [3 H]BIDN binding are 200 to 300 nM in rat brain membranes and 26 to 61 nM in insect membranes. [3 H]BIDN is available commercially (40 to 60 Ci/mmol; Perkin Elmer NEN).
Picrotoxin Site of GABAA Receptors
For [3 H]BIDN binding assays, follow Basic Protocols 1 to 4, replacing [35 S]TBPS with [3 H]BIDN. The assay buffer (50 mM Tris·Cl, pH 7.4) should be adjusted to contain 0.5 M NaCl at all stages. Incubation should be carried out for 45 min at 37°C for rat brain membranes or for 45 min at 24°C for insect membranes. In general, higher incubation temperatures enhance the association kinetics but decrease the affinity (higher Kd value) of the radioligand. Thus, it is important to strike a balance between these two parameters by selecting appropriate incubation conditions to obtain an acceptable binding signal. The above incubation temperatures are recommended when using [3 H]BIDN to obtain optimal binding signals. Nonspecific binding is carried out using unlabeled BIDN (50 µM). If DMSO is used to dissolve compounds, its final concentration should not exceed 0.1% (v/v) in the final 1-ml assay volume.
1.18.12 Supplement 63
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PREPARATION OF RAT BRAIN MEMBRANES Membranes can be prepared either from fresh brain sample or tissue samples that have been stored frozen at −80°C.
SUPPORT PROTOCOL
Materials Brain tissue sample 0.32 M sucrose solution in 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold Potter-Elvehjem glass homogenizer with Teflon pestle Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 50-ml polypropylene and 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) 1. Place fresh/frozen brain tissue into 15 vol ice-cold 0.32 M sucrose solution (pH 7.4) in a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. Allow tissue to thaw if it is frozen. Homogenize tissue until a uniform mixture is obtained and transfer the contents to a 50-ml polypropylene centrifuge tube. Ten to twelve up-and-down strokes are usually sufficient.
2. Centrifuge homogenate 10 min at 1000 × g, 4°C. 3. Discard pellet and transfer the supernatant to a 25-ml polycarbonated centrifuge tubes. Centrifuge supernatant 30 min at 140,000 × g at 4°C to obtain a pellet containing the mitochondrial plus microsomal fraction (i.e., P2 + P3 fraction). 4. Disperse this fraction (pellet) in 15 vol ice-cold double-distilled deionized water. Homogenize in a tissue homogenizer (Polytron or Tissumizer) at midpoint setting for two 10-sec bursts, 10 sec apart. 5. Centrifuge 30 min at 140,000 × g, 4°C, and suspend the pellet in 15 vol ice-cold 50 mM Tris·Cl by homogenization. Multiple washings of the tissue are necessary to remove endogenous GABA that is present in high concentrations in brain tissue; otherwise, residual GABA in the membrane preparation will interfere with the binding assay.
6. Centrifuge and wash as in step 5, and freeze the suspension at −80°C overnight. 7. Thaw the suspension and wash three more times as in step 5. Following the final centrifugation, suspend the pellet in small volume (e.g., 1 ml/g) of ice-cold 50 mM Tris·Cl by homogenization and store frozen in 500-µl aliquots for up to 3 months at −80°C. A freeze-thaw cycle followed by homogenization and centrifugation helps in removing endogenous GABA.
COMMENTARY Background Information The GABAA receptor is a transmembrane hetero-oligomeric pentameric subunit assembly derived from various subunits such as α1 to α6 , β1 to β3 , γ1 to γ3 , δ, ɛ, π, and ρ1 to ρ3 and it is expressed in the peripheral and central nervous systems (Mehta and Ticku, 1999b). The β subunit of GABAA receptors has been suggested as a necessary requirement for the picrotoxin site (Slany et al.,
1995; Zezula et al., 1996). Although the homooligomeric GABAA receptors derived from the β3 -subunit exhibit a specific high-affinity binding for [35 S]TBPS, the GABAA receptor assemblies consisting of α1 β3 γ2 or α1 β3 subunits have higher affinity for [35 S]TBPS over those consisting of β3 γ2 or β3 subunits (Slany et al., 1995; Zezula et al., 1996). GABA produces both pre- and post-synaptic inhibition in a variety of preparations by increasing the
Receptor Binding
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Picrotoxin Site of GABAA Receptors
conductance of chloride ions. One of the first compounds that was shown to block the GABA responses in crayfish was picrotoxin (Elliott and Florey, 1956; Takeuchi and Takeuchi, 1969), which was known to have a convulsant effect as early as 1875. Later studies revealed that picrotoxin antagonizes GABA responses in vertebrates in a noncompetitive manner, suggesting the possibility of different sites of action for these drugs. Picrotoxin is made up of picrotoxinin and picrotin in an equimolar ratio; picrotoxinin is 50-fold more potent than picrotin. To investigate whether there is a distinct site for picrotoxin-like convulsants at the GABA synapse, [3 H]α-dihydropicrotoxinin (DHP) was synthesized and its binding characterized (Ticku et al., 1978; Leeb-Lundberg et al., 1981). These studies revealed that the specific binding to brain membranes was only 10% to 15% of the total binding, and it was inhibited by GABAergic drugs, suggesting that the picrotoxin site at the GABA synapse is a target of drug action for several categories of centrally acting agents that affect GABAergic transmission (Ticku et al., 1978; LeebLundberg et al., 1981). Subsequently, other radioligands, such as [35 S]TBPS (or [35 S]TBPT), [3 H]TBOB, [3 H]EBOB, and [3 H]BIDN, were introduced to study the picrotoxin site of the GABAA receptor. The greatest advantage of [35 S]TBPS over [3 H]DHP is a far better signal-to-noise ratio. The binding of [35 S]TBPS to brain membranes is specific, saturable, chlorideion dependent, and modulated by GABAergic drugs (Squires et al., 1983; Ramanjaneyulu and Ticku, 1984a,b; Supavilai and Karobath, 1984; Mehta and Ticku, 1998, 1999a). Its binding to brain membranes is inhibited by picrotoxin, pentylenetetrazole, and other tetrazoles in a competitive manner (Ramanjaneyulu and Ticku, 1984a,b; Supavilai and Karobath, 1984; Mehta and Ticku, 1998, 1999a). Agonists of the benzodiazepine site enhance, whereas inverse benzodiazepine agonists inhibit [35 S]TBPS binding (Supavilai and Karobath, 1984). Muscimol, pentobarbital, and etazolate elicit biphasic effects on [35 S]TBPS binding, suggesting that these drugs interact differentially with two populations of the picrotoxin site on GABAA receptors (Supavilai and Karobath, 1984). [3 H]TBOB and [35 S]TBPS bind to the identical picrotoxin site of the GABAA receptor (Lawrence et al., 1985). However, [3 H]TBOB has a much longer radioactive half-life and binds to rat brain membranes at 0°, 24°,
and 37°C with similar Kd values, whereas [35 S]TBPS has negligible binding at 0°C. Furthermore, a shorter incubation time is required for [3 H]TBOB binding (90 versus 180 min at 24°C). In spite of these advantages, [35 S]TBPS is more commonly used as a research tool due to its lower Kd value at 24°C (20 nM versus 60 nM; Lawrence et al., 1985; Casida and Lawrence, 1985), and also because [3 H]TBOB binding is considerably less sensitive to inhibition by GABA, muscimol, and Ro 5-4864 as compared to [35 S]TBPS binding, due to differences in the nature of interactions of these radioligands with the identical picrotoxin site of the GABAA receptor (Squires et al., 1983; Ticku and Ramanjaneyulu, 1984; Lawrence et al., 1985). However, [3 H]TBOB offers the advantage of superior resolution in autoradiographic studies. [3 H]EBOB is structurally related to 3 [ H]TBOB and has high affinity (Kd 2 nM) for the picrotoxin site on the GABAA receptor and in contrast to [35 S]TBPS, also binds to invertebrate GABAA receptors (Cole and Casida, 1992; Hawkinson and Casida, 1992; Yagle et al., 2003). [3 H]BIDN is suitable for studies of GABAgated chloride channels of vertebrates as well as insects (Rauh et al., 1997). The cage convulsants (EBOB, TBPS, and TBOB) and picrotoxinin displace [3 H]BIDN binding in rat membranes but not in insect membranes, indicating that the convulsant site is somewhat different in insects (Rauh et al., 1997). However, dieldrin displaces [3 H]BIDN binding competitively in insect membranes, suggesting that the sites of action of picrotoxinin and dieldrin are not identical. Unlike picrotoxin, which antagonizes a variety of ligand-gated chloride channels, such as vertebrate glycine-gated and invertebrate L-glutamate-gated chloride channels, BIDN blocks only GABA-gated chloride channels in vertebrates and insects (Hamon et al., 1998). Kd values for [3 H]BIDN binding are 200 to 300 nM in rat brain membranes and 26 to 61 nM in insect membranes. [3 H]BIDN is not a very popular tool for studies involving rat brain membranes because of its lower affinity (higher Kd ) compared to other radioligands. Scatchard analysis and dissociation studies using various radioligands for the picrotoxin site, followed by analysis of the binding data using an iterative fitting computer program (e.g., LIGAND; Munson and Rodbard, 1980) or linear regression analysis, are utilized to investigate whether GABAergic drugs bind to the picrotoxin site or modulate this site as a result of their binding
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Table 1.18.1 IC50 and Ki Values of Various Ligands for [35 S]TBPS Binding in Rat Brain Membranesa
IC50 (mM)
Ki (µM)
Anisatin
0.10
0.09
Bemegride
200
185
6-o-Chlorophenyltetrazole
310
287
1-Cyclohexyl-5-methyl-tetrazole
40
37
6, 6 -Dichloropentamethylenetetrazole
10
9.3
Ethylbicyclophosphate
1.2
1.1
Ethyl-β–carboline-3-carboxylate (βCCE)
>2000
>2000
Heptamethylenetetrazole
230
213
1-Isobutyl-5-methylenetetrazole
480
444
Isopropylbicyclophosphate
0.16
0.15
L-Kynurenine
>1000
>1000
N-Methyl-β-carboline-carboxamide (FG7142)
>2000
>2000
1-Methyl-5-cyclohexyltetrazole
500
463
Methyl-6, 7-dimethoxy-4-ethyl-β-carboline-3 -carboxylate (DMCM)
>1000
>1000
7-Methyl-9-isopropylpentamethylenetetrazole
200
185
7-Methyl-10-isopropylpentamethylenetetrazole
145
134
7-Methylpentamethylenetetrazole
160
148
Pentylenetetrazole (PTZ)
700
648
Picrotoxinin
0.40
0.37
Quinolinic acid
120
111
Ro 5-3663
22
20
Buspirone HCl
>2000
>2000
γ-Butyrolactone
>2000
>2000
Cannabidiol
40
37
Cartazolate
0.52
0.48
Dextronantradol HCl
50
46
Diazepam
>200
>200
(±)5-(1,3-dimethylbutyl)-5-ethyl barbituric acid (DMBB)
50
46
Etazolate
5
4.6
Ethanol
500
463
(–)Etomidate
100
93
(+)Etomidate sulfate
9
8.3
Flurazepam
>200
>200
GABA
0.45
0.42
(–)Hexobarbital
380
352
(+)Hexobarbital
140
130
Ligand Convulsants
sulfate
Depressants, anticonvulsants, anxiolytics, and other drugs:
continued
Receptor Binding
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Table 1.18.1 IC50 and Ki Values of Various Ligands for [35 S]TBPS Binding in Rat Brain Membranesa , continued
Ligand
IC50 (mM)
Ki (µM)
(±)Hexobarbital
220
204
(–)Ketamine HCl
>2000
>2000
(+)Ketamine HCl
>2000
>2000
Levonantradol HCl
40
37
(±)Mephobarbital
210
194
Methaqualone
100
93
Metharbital
145
134
(–)N-Methyl-5-phenylpropyl barbituric acid (MPPB)
116
107
(+)N-Methyl-5-phenylpropyl barbituric acid (MPPB)
760
704
MK-801
>400
>400
(–)Pentobarbital
70
65
(+)Pentobarbital
125
116
(±)Pentobarbital
90
83
Phenobarbital
480
444
Ro 5-4864
10
9.3
Ro 11-6896
16
14.8
(–)Secobarbital
40
37
(+)Secobarbital
70
65
(±)Secobarbital
20
18.5
Tofizopam
160
148
a Drugs that inhibit specific [35 S]TBPS binding by
UNIT 1.18
Ashok K. Mehta1 and Maharaj K. Ticku1 1
The University of Texas Health Science Center, San Antonio, Texas
ABSTRACT This unit describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd , Bmax determination), association kinetics, dissociation kinetics, and competition assays (IC50 , Ki determination) are included. Curr. Protoc. C 2013 by John Wiley & Sons, Inc. Pharmacol. 63:1.18.1-1.18.18. Keywords: GABAA receptor r picrotoxin binding site r allosteric regulation r convulsants
INTRODUCTION NOTE: This unit was updated by the editors of Current Protocols in Pharmacology in 2013 to document changes related to the subject matter since the original publication and also to reflect current “state of the art” reference compounds, suppliers, and literature citations. This unit describes an in vitro assay for characterization of the picrotoxin site of γ-aminobutyric acid A (GABAA ) receptors in rat brain membranes using various radioligands, such as [35 S]t-butylbicyclophosphorothionate (35 STBPS or [35 S]TBPT), [3 H]t-butylbicycloorthobenzoate([3 H]TBOB), 4 -ethynyl-4-n-[2,3-3 H2 ]propylbicycloorthobenzoate ([3H]EBOB), and [3 H]3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3 H]BIDN). GABAA receptors are transmembrane hetero-oligomeric pentameric subunit assemblies derived from various subunits (such as α1 to α6 , β1 to β3 , γ1 to γ3 , δ, ɛ, π, and ρ1 to ρ3 ), and the β-subunit of GABAA receptors has been suggested as a necessary requirement for the picrotoxin site (Slany et al., 1995; Zezula et al., 1996). The four Basic Protocols describe procedures for investigating the picrotoxin site of GABAA receptors and its interaction with various compounds using [35 S]TBPS as a radioligand. Three Alternate Protocols describe the use of [3 H]TBOB (Alternate Protocol 1), [3 H]EBOB (Alternate Protocol 2), and [3 H]BIDN (Alternate Protocol 3). Preparation of the membrane suspension for this assay is described in a Support Protocol.
Update There has been a resurgence of interest in the GABAA receptor picrotoxin binding site with the growing interest in discovering and developing allosteric, as opposed to orthosteric, GABA receptor agonists and antagonists. The protocols detailed in this unit describing binding assays for characterizing the picrotoxin binding site remain current. The ligand of choice for labeling this site is still [35 S]TBPS, although [3 H]TBOB and [3 H]EBOB are occasionally employed as well. As with most binding assays, all of these protocols can be conducted using multi-well microplates instead of a cell harvester. CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area, following the guidelines provided by the local radiation safety officer. Current Protocols in Pharmacology 1.18.1-1.18.18, December 2013 Published online December 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471141755.ph0118s63 C 2013 John Wiley & Sons, Inc. Copyright
Receptor Binding
1.18.1 Supplement 63
BASIC PROTOCOL 1
SATURATION BINDING OF [35 S]TBPS TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES This protocol describes an in vitro assay for labeling the picrotoxin site of GABAA receptors in rat brain membranes using [35 S]TBPS as the radioligand. It has also been shown that this ligand can be employed to label GABAA receptors in post-mortem brain tissue, although the binding site in human tissue is more labile to degradation than that of rat (Atack et al., 2007). Picrotoxinin is used to define nonspecific binding. This protocol can be utilized to determine the Kd (affinity) of radioligands in saturation binding experiments (see UNIT 1.3). It is advisable to use approximately ten concentrations of radioligand ranging from 10-fold below to 20-fold above the estimated Kd value. The final volume of the binding assays for routine experiments is 1 ml, but it can be scaled to 0.5 ml or 2.0 ml, depending on the cost and desired accuracy. Pharmaceutical companies even use 100- or 200-µl assays for routine high-throughput screening.
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl, pH 7.4 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN Life) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester) Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer data graphing/fitting program such as LIGAND; Munson and Rodbard, 1980, or Prism (GraphPad) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: Picrotoxinin should first be dissolved to 100 mM in dimethylsulfoxide (DMSO), but the final concentration of DMSO should not exceed 0.1% (v/v) in the 1-ml assay.
Prepare membrane suspension for binding assay 1. On the day of the assay, thaw a frozen membrane preparation and resuspend in 20 to 50 vol ice-cold 50 mM Tris·Cl in a 25-ml centrifuge tube using a tissue homogenizer (midpoint setting). 2. Centrifuge homogenate 30 min at 140,000 × g, 4°C. 3. Discard supernatant, suspend resultant pellet in ice-cold 50 mM Tris·Cl, and centrifuge 30 min at 140,000 × g, 4°C. Repeat this step one more time. Multiple washings of the tissue are necessary to remove endogenous GABA that is present in high concentrations in brain tissue; otherwise, residual GABA in the membrane preparation will interfere with the binding assay. However, it is adequate to wash the membrane suspension two times on the day of assay (omitting the third wash) for preliminary results/screening, provided the membrane is prepared over a 2-day period using the freeze-thaw cycle described (see Support Protocol). Picrotoxin Site of GABAA Receptors
4. Resuspend the final pellet in ice-cold 50 mM Tris·Cl to yield a protein concentration of 2 to 3 mg/ml.
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One gram of tissue yields 20 mg protein. Protein concentration may be measured using BCA (Pierce), Lowry, Bradford, or other suitable assay with BSA as reference standard (or γ -globulin in the case of the Bradford assay). See APPENDIX 3A for protein assays. The final protein content in the 1.0-ml assay mixture should be in the range of 0.2 to 0.3 mg.
Generate binding-site saturation data 5. To measure total binding: Prepare triplicate 12 × 75–mm borosilicate glass culture tubes for total binding by combining the following reagents, using ten concentrations of radioligand in the range indicated. 100 µl 1.5 M KCl (150 mM final) 0.4 to 100 µl 1.2 µM [35 S]TBPS (0.5 to 120 nM final) 50 mM Tris·Cl to 0.9 ml. Saturation data are obtained using increasing concentrations of the radioligand in the absence (total binding) and presence (blank; see step 6) of a saturating concentration of displacer (100 µM picrotoxinin). Total and nonspecific binding are determined in parallel for each concentration of radioligand. It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent. In the case of [35 S]TBPS (Kd 20 nM), a concentration range of 0.5 to 120 nM yields reliable and reproducible saturation kinetic data. In general, ten concentrations of a radioligand covering a range from 10-fold below to 20-fold above its Kd value are usually sufficient for saturation binding assays. To investigate the saturation binding kinetics of a test compound, replace [35 S]TBPS with a radiolabeled test compound using concentrations in this range.
6. To measure nonspecific binding: Prepare parallel triplicate tubes for nonspecific binding at the same radioligand concentrations. Combine reagents as described in step 5, but add 100 µl of 1 mM picrotoxinin (final 100 µM) and decrease the Tris·Cl to maintain a 0.9-ml volume. 7. To initiate the binding assay, add 100 µl membrane suspension (0.2 to 0.3 mg protein) to each assay tube (final 1.0 ml), gently vortex to mix the contents, and incubate 180 min at room temperature (24°C) to achieve binding equilibrium. [35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
8. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper with polyethyleneimine (PEI).
9. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
10. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 11. Calculate the specific radioligand binding (dpm) at each radioligand concentration by subtracting nonspecific binding from total binding. Receptor Binding
1.18.3 Current Protocols in Pharmacology
Supplement 63
120
1.0
80
Bound/free (× 10 –6)
Specific [35S]TBPS binding (pmol/mg protein)
1.5
0.5
0
40
0 0
10
20
30
40
[35S] TBPS (nM)
0
1.
.0
Bound (pmol/mg protein)
C 100 picrotoxinin (+) etomidate (–) etomidate Specific [35S] TBPS binding
80
60
40
20
0 10 –9
10 –8
10 –7
10 –6 [Ligand] (M)
10 –5
10 –4
10 –3
Figure 1.18.1 [35 S]TBPS binding to rat brain membranes. (A) Saturation of specific [35 S]TBPS binding with increasing concentrations of [35 S]TBPS in the absence and presence of picrotoxinin (100 µM). (B) Scatchard plot of specific [35 S]TBPS binding from panel A. Kd and Bmax values were found to be 25 nM and 1.5 pmol/mg protein, respectively. (C) Concentration-dependent inhibition of specific [35 S]TBPS binding by picrotoxinin and stereoisomers of etomidate in rat brain membranes. IC50 values of picrotoxinin, (+)etomidate, and (–)etomidate were found to be 0.4 µM, 9 µM, and 100 µM, respectively. Reprinted from Ramanjaneyulu and Ticku (1984a) with permission from Raven Press.
Picrotoxin Site of GABAA Receptors
12. Convert specific binding to the concentration of radioligand bound. Use a Scatchard transformation plot (bound ligand versus bound/free ligand) to determine the affinity constant of the radioligand (Kd ) and the maximum number of binding sites (Bmax ) by using a suitable iterative fitting computer program, such as LIGAND
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(Munson and Rodbard, 1980) or Prism (GraphPad) or linear regression analysis (see Fig. 1.18.1A,B). See UNIT 1.3 for a thorough explanation of the calculations. For a detailed procedure to analyze the data using computer programs, the experimenter is advised to refer to the program manuals. Kd values can also be derived from the association and dissociation kinetics data as described below (see Basic Protocols 3 and 4).
COMPETITION ASSAYS FOR [35 S]TBPS BINDING TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES
BASIC PROTOCOL 2
This protocol can be utilized to determine the potency (IC50 or Ki ) of unlabeled test compounds in competition experiments. This procedure requires an unlabeled test compound or reference compound, a radioligand (e.g., 4 nM [35 S]TBPS for labeling picrotoxin sites), and a reference displacer (e.g., 100 µM picrotoxinin) to define nonspecific binding by using a concentration sufficient to completely displace the radioligand from the receptors. For determining the IC50 or Ki value of a test compound, ten concentrations of the unlabeled compound, covering a range from 10-fold below to 20-fold above its estimated IC50 value, are usually sufficient to determine reliable and reproducible values. Furthermore, the radioligand concentration should be less than the Kd value of the radioisotope (approximately one-tenth to one-half of the Kd value, which is 20 nM for [35 S]TBPS).
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl, pH 7.4 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN) in 50 mM Tris·Cl, pH 7.4 Unlabeled competitor (test compound) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester) Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer program (e.g., DeltaGraph; Delta Point) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: If unlabeled competitor is insoluble in 50 mM Tris·Cl, dimethylsulfoxide (DMSO) can be added. Picrotoxinin should first be dissolved to 100 mM in DMSO, but in all cases, the final concentration of DMSO should not exceed 0.1% (v/v) in the final 1-ml assay volume.
Prepare membrane suspension for binding assay 1. Prepare membranes as described (see Basic Protocol 1, steps 1 to 4). Generate competitive binding data 2. To measure total binding: Prepare a set of triplicate 12 × 75–mm borosilicate glass culture tubes for total binding in the absence (zero concentration of competitor)
Receptor Binding
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Supplement 63
and increasing concentrations of unlabeled competitor by combining the following reagents:
100 µl 1.5 M KCl (150 mM final) 3.3 µl 1.2 µM [35 S]TBPS (4 nM final) Various concentrations of unlabeled competitor, including zero 50 mM Tris·Cl to 0.9 ml. Use ten concentrations of unlabeled competitor in a range from 10-fold below to 20-fold above its estimated IC50 or Ki . It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent.
3. To measure nonspecific binding: Prepare a set of parallel triplicate tubes for nonspecific binding. Combine reagents as described in step 2, but replace the unlabeled competitor with 100 µl of 1 mM picrotoxinin (final 100 µM). 4. To initiate the binding assay, add 100 µl membrane suspension (0.2 to 0.3 mg protein) to each assay tube (final 1.0 ml), gently vortex to mix the contents, and incubate 180 min at room temperature (24°C) to achieve binding equilibrium. [35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
5. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper with polyethyleneimine (PEI).
6. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
7. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 8. Calculate the specific radioligand binding (dpm) at each concentration of competitor by subtracting nonspecific binding from total binding. 9. Calculate the percentage inhibition of the specific radioligand binding by each concentration of competitor. Plot the percentage inhibition versus competitor concentration, and calculate the concentration that inhibits 50% of the specific radioligand binding (IC50 ) using a suitable computer program such as DeltaGraph. Calculate the inhibitory constant (Ki ) using the equation: Ki = IC50 /(1 + [L]/Kd ), where [L] is the molar concentration of radioligand and Kd is the affinity constant. See Figure 1.18.1C for a representative plot. Caution should be exercised in setting the limits for various variables while analyzing the data using such computer programs, because varied results may be obtained if unrealistic values are set. BASIC PROTOCOL 3 Picrotoxin Site of GABAA Receptors
ASSOCIATION KINETICS OF BINDING TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES This protocol describes an in vitro assay for determining the association kinetics of binding (association rate constant and affinity constant) of various radioligands to the
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picrotoxin site of GABAA receptors in rat brain membranes. These studies are useful in investigating the pharmacological interaction of GABAergic drugs with the picrotoxin site.
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl, pH 7.4 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN) in 50 mM Tris·Cl, pH 7.4 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester) Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer program (e.g., DeltaGraph; Delta Point) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: Picrotoxinin should first be dissolved to 100 mM in dimethylsulfoxide (DMSO), but the final concentration of DMSO should not exceed 0.1% (v/v) in the 1-ml assay.
Prepare membrane suspension for binding assay 1. Prepare membranes as described (see Basic Protocol 1, steps 1 to 4). Generate association kinetics data 2. To measure total binding: Prepare triplicate 12 × 75–mm borosilicate glass culture tubes for total binding for each time interval by combining the following reagents: 100 µl 1.5 M KCl (150 mM final) 3.3 µl 1.2 µM [35 S]TBPS (4 nM final) 50 mM Tris·Cl to 0.9 ml. For association kinetics, data are obtained in the absence (total binding) and presence (nonspecific binding) of a saturating concentration of displacer (100 µM picrotoxinin) using multiple incubation times. Total and nonspecific binding are determined in parallel at each time interval using a single concentration of [35 S]TBPS (4 nM final). It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent. To investigate the association kinetics of a test compound, replace [35 S]TBPS with a radiolabeled test compound. The optimum concentration should be determined using Basic Protocol 1.
3. To measure nonspecific binding: Prepare parallel triplicate tubes for nonspecific binding for each time interval. Combine reagents as described in step 2, but add 100 µl of 1 mM picrotoxinin (final 100 µM) and decrease the Tris·Cl to maintain a 0.9-ml volume. 4. To initiate the binding assay, add 100 µl membrane suspension (0.2 to 0.3 mg protein) to each assay tube (final 1.0 ml), gently vortex to mix the contents, and incubate corresponding nonspecific and total binding tubes for different periods of time (e.g., 10, 20, 40, 60, 90, 120, and 180 min) at room temperature. Current Protocols in Pharmacology
Receptor Binding
1.18.7 Supplement 63
A
Be–B t (%) Be
100 90 80 70 60 50
40 30 20
10 20
80
40 60 Time (min)
100
B 100
B t/Be (%)
50
10 5 6 mM PTZ 10 µM GABA 100 µM etazolate
1 0
10
20
30
40
50
60
70
80
90
100
Time (min) Figure 1.18.2 Analysis of association and dissociation kinetics of [35 S]TBPS binding to rat cortical membranes. (A) Association of [35 S]TBPS (4 nM) to rat cortical membranes at 25°C. The apparent association rate constant (kapp ) and association rate constant (k1 ) of [35 S]TBPS were found to be 0.0183 min–1 and 3.6 × 106 min–1 M–1 , respectively. (B) Dissociation of [35 S]TBPS (3 nM) binding from rat cortical membranes by pentylenetetrazole (PTZ; 6 mM), GABA (10 µM), and etazolate (100 µM) at 25°C. The respective half-lives of dissociation with these drugs were found to be 68 min, 1.3 min (first phase)/12 min (second phase), and 0.7 min (first phase)/20 min (second phase). Reprinted from Maksay and Ticku (1985) with permission from Raven Press.
[35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
Picrotoxin Site of GABAA Receptors
Execute the timing of the assay assembly in such a way that the termination of binding reaction in the assay tubes can be completed in a short time without any significant interruption in the filtration process. It usually takes 2 to 3 min to process one rack of 48 assay tubes using a vacuum filtration device with 48 slots (e.g., Brandel cell harvester model MB-48L). Therefore, a gap of 4 to 5 min should be maintained between different racks of assay tubes. The time course must include a 180-min time point so that Be (step 9) can be calculated.
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5. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper with polyethyleneimine (PEI).
6. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
7. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 8. Calculate the specific radioligand binding (dpm) by subtracting nonspecific binding from total binding. 9. Plot ln[(Be – Bt )/Be ] against time t, where Be and Bt are specific binding at equilibrium and at time t, respectively (see Fig. 1.18.2A). Calculate the apparent association rate constant (kapp ) from the slope of the resulting line. 10. Determine the association rate constant (k1 ) using the equation kapp = k1 [L] + k–1 , where [L] is the molar concentration of radioligand and k–1 is the dissociation rate constant (see Basic Protocol 4). Any suitable computer program such as DeltaGraph can be used for analyzing the association kinetic data. However, caution should be exercised in setting the limits for various variables while analyzing the data using such computer programs because varied results may be obtained if unrealistic values are set.
DISSOCIATION KINETICS OF BINDING TO THE PICROTOXIN SITE OF GABAA RECEPTORS IN RAT BRAIN MEMBRANES
BASIC PROTOCOL 4
This protocol describes an in vitro assay for determining the dissociation kinetics of binding (dissociation rate constant and affinity constant) of various radioligands to the picrotoxin site of GABAA receptors in rat brain membranes. These studies are utilized to investigate whether GABAergic drugs bind to the picrotoxin site or modulate this site as a result of their binding to a distinct, but allosterically coupled, site on the GABAA receptor.
Materials Frozen membrane preparation (see Support Protocol) 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 1.5 M KCl in 50 mM Tris·Cl 1.2 µM [35 S]t-butylbicyclophosphorothionate ([35 S]TBPS; >60 Ci/mmol; Perkin Elmer NEN) in 50 mM Tris·Cl 1 mM picrotoxinin (Sigma) in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Unlabeled test compound in 50 mM Tris·Cl, pH 7.4 (see NOTE below) Scintillation cocktail 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 12 × 75–mm borosilicate glass culture tubes Whatman GF/B glass-fiber filters Vacuum filtration device (e.g., Brandel cell harvester)
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Filter forceps (Millipore) 6-ml scintillation vials Liquid scintillation counter Computer program (e.g., DeltaGraph; Delta Point) Additional reagents and equipment for protein assays (see APPENDIX 3A) NOTE: If unlabeled competitor is insoluble in 50 mM Tris·Cl, dimethylsulfoxide (DMSO) can be added. Picrotoxinin should first be dissolved to 100 mM in DMSO, but in all cases, the final concentration should not exceed 0.1% (v/v) in the final 1-ml assay volume.
Prepare membrane suspension for binding assay 1. Prepare membranes as described (see Basic Protocol 1, steps 1 to 4). Generate dissociation kinetics data 2. To measure total binding: Prepare and label triplicate 12 × 75–mm borosilicate glass culture tubes for total binding and add the following reagents: 100 µl 1.5 M KCl (150 mM final) 3.3 µl 1.2 µM [35 S]TBPS (4 nM final) 50 mM Tris·Cl to 0.9 ml. For dissociation kinetics, equilibrium data are obtained in the absence (total binding) and presence (nonspecific binding) of a saturating concentration of displacer (100 µM picrotoxinin), and binding data are obtained for various time points in the presence of a saturating concentration of an unlabeled test compound (displacer) that causes dissociation of the bound radioligand. Total and nonspecific binding are determined in parallel using a single concentration of [35 S]TBPS (4 nM final). The total assay volume will be 1 ml after the addition of membrane suspension (step 5). It is important to add KCl (150 mM final), because [35 S]TBPS binding is Cl– ion dependent. To investigate the dissociation kinetics of a test compound, replace [35 S]TBPS with a radiolabeled test compound. The optimum concentration should be determined using Basic Protocol 1.
3. To measure nonspecific binding: Prepare triplicate tubes for nonspecific binding. Combine reagents as described in step 2, but add 100 µl of 1 mM picrotoxinin (final 100 µM) and decrease the Tris·Cl to maintain the appropriate volume. 4. To measure dissociation kinetics: For each time interval (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 60, 80, and 100 min), prepare triplicate tubes as in step 2, but decrease Tris·Cl to allow for addition of unlabeled test compound (step 6). 5. Initiate the binding assay in all tubes by adding 100 µl membrane suspension (0.2 to 0.3 mg protein). Gently vortex to mix the contents, and begin to incubate at room temperature. [35 S]TBPS has negligible binding at 0°C (Squires et al., 1983).
6. Initiate the dissociation of [35 S]TBPS binding at the appropriate times by adding a saturating concentration of unlabeled test compound (displacer) to the tubes prepared in step 4. Vortex gently. Continue to incubate all tubes for a total of 180 min.
Picrotoxin Site of GABAA Receptors
Time the addition of displacer such that the membrane suspension is incubated for 180 min with [35 S]TBPS and for a specified time with a displacer, and so that all the tubes, including those for total and nonspecific binding, are ready for the termination
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of the binding reaction without any significant interruption in the filtration process. For example, in order to obtain the data point representing the dissociation of the [35 S]TBPS binding in 100 min, add membrane suspension to the tubes containing buffer, KCl, and [35 S]TBPS (with and without picrotoxinin). Vortex the tubes gently. Wait for 80 min, and then add displacer. Vortex the tubes gently, incubate an additional 100 min, and then terminate the binding reaction.
7. Terminate the binding reaction by filtering the contents of the test tubes through Whatman GF/B glass-fiber filters maintained under reduced pressure in a vacuum filtration device. Wash each filter rapidly three times (3 to 5 sec each) with 2 ml ice-cold 50 mM Tris·Cl. The washing time should be kept constant and as short as possible so as to minimize additional dissociation of bound radioligand from the receptors. It is not necessary to soak the filter paper in polyethyleneimine (PEI).
8. Transfer each filter to a 6-ml scintillation vial using filter forceps, add 4 ml scintillation cocktail, and allow to sit for 12 hr at room temperature. Shaking the vials can reduce this period.
9. Quantify radioactivity using a liquid scintillation counter.
Analyze binding data 10. Calculate the specific radioligand binding (dpm) by subtracting nonspecific binding from total binding (Be ) and from each time interval (Bt ). 11. Plot ln[Bt /Be ] versus time t, where Bt and Be are the amount of specific binding at time t and at equilibrium, respectively. Calculate the rate constant of dissociation (k–1 ) from the slope of the resulting line (see Fig. 1.18.2B). Any suitable computer program such as DeltaGraph can be used for analyzing the dissociation kinetic data. However, caution should be exercised in setting the limits for various variables while analyzing the data using such computer programs because varied results may be obtained if unrealistic values are set.
12. Calculate the affinity constant (Kd ) of the radioligand by dividing the dissociation rate constant (k–1 ) by the association rate constant (k1 ).
CHARACTERIZATION OF THE PICROTOXIN SITE OF GABAA RECEPTORS USING [3 H]TBOB
ALTERNATE PROTOCOL 1
[3 H]t-Butylbicycloorthobenzoate ([3 H]TBOB) and [35 S]TBPS label the same picrotoxin site on the GABAA receptor (Lawrence et al., 1985). However, [3 H]TBOB has a much longer radioactive half-life, and binding assays conducted at 0°C, 24°C, and 37°C yield similar Kd values, whereas [35 S]TBPS has negligible binding at 0°C. Furthermore, a shorter incubation time is needed for [3 H]TBOB binding (90 min versus 180 min at 24°C). In spite of these advantages, [35 S]TBPS is more commonly used as a research tool due to its relatively lower Kd value (20 nM versus 60 nM) at 24°C, and because [3 H]TBOB binding is considerably less sensitive than [35 S]TBPS binding to inhibition by GABA, muscimol, and Ro 5-4864, due to differences in the interactions of these radioligands with the identical picrotoxin site of the GABAA receptor (Squires et al., 1983; Ticku and Ramanjaneyulu, 1984; Lawrence et al., 1985). However, [3 H]TBOB is a superior radioligand for autoradiographic studies, due to better resolution. [3 H]TBOB is available commercially (20 to 60 Ci/mmol; GE Healthcare Life Sciences; PE NEN Life Sciences). For [3 H]TBOB binding assays, follow Basic Protocols 1 to 4, replacing [35 S]TBPS with [3 H]TBOB. The assay buffer (50 mM Tris·Cl, pH 7.4) should be adjusted to contain
Receptor Binding
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0.5 M NaCl at all stages. Incubation should be carried out for either 90 min at 24°C, 120 min at 0°C, or 60 min at 37°C. Nonspecific binding can be carried out using picrotoxinin (100 µM) or unlabeled TBOB (4 µM). Dimethylsulfoxide (DMSO) is used to dissolve picrotoxinin as well as TBOB, with a final concentration of DMSO not exceeding 0.1% (v/v) in the final 1-ml assay volume. ALTERNATE PROTOCOL 2
CHARACTERIZATION OF THE PICROTOXIN SITE OF GABAA RECEPTORS USING [3 H]EBOB 4 -Ethynyl-4-n-[2,3-3 H2 ]propylbicycloorthobenzoate ([3 H]EBOB), also called [3 H]ethynylbicycloorthobenzoate, is structurally related to TBOB. It has high affinity (Kd 2 nM) for the picrotoxin site on the GABAA receptor and is available commercially (30 to 60 Ci/mmol; Perkin Elmer NEN). Besides having a longer radioactive half-life and higher affinity than [35 S]TBPS, [3 H]EBOB binds to both vertebrate and invertebrate GABAA receptors (Yagle et al., 2003). For [3 H]EBOB binding assays, follow Basic Protocols 1 to 4, replacing [35 S]TBPS by [3 H]EBOB. The assay buffer (50 mM Tris·Cl, pH 7.4) should be adjusted to contain 0.3 M NaCl at all stages. Incubation should be carried out for 120 min at 24°C or 90 min at 37°C. Nonspecific binding can be carried out using picrotoxinin (100 µM), unlabeled EBOB (2 µM), or lindane (5 µM; Sigma). If DMSO is used to dissolve compounds, its final concentration should not exceed 0.1% (v/v) in the final 1-ml assay volume.
ALTERNATE PROTOCOL 3
CHARACTERIZATION OF PICROTOXIN SITE OF GABAA RECEPTOR USING [3 H]BIDN [3 H]3,3-bis-Trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3 H]BIDN) is suitable for studies of GABA-gated chloride channels of vertebrates and insects. The cage convulsants (TBPS, and TBOB) and picrotoxinin displace [3 H]BIDN binding in rat membranes, but not in insect membranes, suggesting that the convulsant site of the GABAA receptor is somewhat different in insects as compared to rats. However, dieldrin displaces [3 H]BIDN binding competitively in insect membranes. Unlike picrotoxin, which antagonizes a variety of ligand-gated chloride channels such as vertebrate glycine-gated and invertebrate L-glutamate-gated chloride channels, BIDN blocks only GABA-gated chloride channels in vertebrates and insects (Rauh et al., 1997). Thus, [3 H]BIDN offers the advantage of being selective for GABA-gated chloride channels, and can be utilized to investigate the pharmacology of chloride channels in vertebrates as well as in insects (Rauh et al., 1997; Hamon et al., 1998). However, its low affinity (higher Kd value) for rat brain membranes limits its use as an experimental tool. Kd values for [3 H]BIDN binding are 200 to 300 nM in rat brain membranes and 26 to 61 nM in insect membranes. [3 H]BIDN is available commercially (40 to 60 Ci/mmol; Perkin Elmer NEN).
Picrotoxin Site of GABAA Receptors
For [3 H]BIDN binding assays, follow Basic Protocols 1 to 4, replacing [35 S]TBPS with [3 H]BIDN. The assay buffer (50 mM Tris·Cl, pH 7.4) should be adjusted to contain 0.5 M NaCl at all stages. Incubation should be carried out for 45 min at 37°C for rat brain membranes or for 45 min at 24°C for insect membranes. In general, higher incubation temperatures enhance the association kinetics but decrease the affinity (higher Kd value) of the radioligand. Thus, it is important to strike a balance between these two parameters by selecting appropriate incubation conditions to obtain an acceptable binding signal. The above incubation temperatures are recommended when using [3 H]BIDN to obtain optimal binding signals. Nonspecific binding is carried out using unlabeled BIDN (50 µM). If DMSO is used to dissolve compounds, its final concentration should not exceed 0.1% (v/v) in the final 1-ml assay volume.
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PREPARATION OF RAT BRAIN MEMBRANES Membranes can be prepared either from fresh brain sample or tissue samples that have been stored frozen at −80°C.
SUPPORT PROTOCOL
Materials Brain tissue sample 0.32 M sucrose solution in 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold 50 mM Tris·Cl, pH 7.4 (APPENDIX 2A), ice cold Potter-Elvehjem glass homogenizer with Teflon pestle Tissue homogenizer (e.g., Brinkmann Polytron or Tekmar Tissumizer) 50-ml polypropylene and 25-ml polycarbonate centrifuge tubes (Beckman or equivalent) 1. Place fresh/frozen brain tissue into 15 vol ice-cold 0.32 M sucrose solution (pH 7.4) in a Potter-Elvehjem glass homogenizer fitted with a Teflon pestle. Allow tissue to thaw if it is frozen. Homogenize tissue until a uniform mixture is obtained and transfer the contents to a 50-ml polypropylene centrifuge tube. Ten to twelve up-and-down strokes are usually sufficient.
2. Centrifuge homogenate 10 min at 1000 × g, 4°C. 3. Discard pellet and transfer the supernatant to a 25-ml polycarbonated centrifuge tubes. Centrifuge supernatant 30 min at 140,000 × g at 4°C to obtain a pellet containing the mitochondrial plus microsomal fraction (i.e., P2 + P3 fraction). 4. Disperse this fraction (pellet) in 15 vol ice-cold double-distilled deionized water. Homogenize in a tissue homogenizer (Polytron or Tissumizer) at midpoint setting for two 10-sec bursts, 10 sec apart. 5. Centrifuge 30 min at 140,000 × g, 4°C, and suspend the pellet in 15 vol ice-cold 50 mM Tris·Cl by homogenization. Multiple washings of the tissue are necessary to remove endogenous GABA that is present in high concentrations in brain tissue; otherwise, residual GABA in the membrane preparation will interfere with the binding assay.
6. Centrifuge and wash as in step 5, and freeze the suspension at −80°C overnight. 7. Thaw the suspension and wash three more times as in step 5. Following the final centrifugation, suspend the pellet in small volume (e.g., 1 ml/g) of ice-cold 50 mM Tris·Cl by homogenization and store frozen in 500-µl aliquots for up to 3 months at −80°C. A freeze-thaw cycle followed by homogenization and centrifugation helps in removing endogenous GABA.
COMMENTARY Background Information The GABAA receptor is a transmembrane hetero-oligomeric pentameric subunit assembly derived from various subunits such as α1 to α6 , β1 to β3 , γ1 to γ3 , δ, ɛ, π, and ρ1 to ρ3 and it is expressed in the peripheral and central nervous systems (Mehta and Ticku, 1999b). The β subunit of GABAA receptors has been suggested as a necessary requirement for the picrotoxin site (Slany et al.,
1995; Zezula et al., 1996). Although the homooligomeric GABAA receptors derived from the β3 -subunit exhibit a specific high-affinity binding for [35 S]TBPS, the GABAA receptor assemblies consisting of α1 β3 γ2 or α1 β3 subunits have higher affinity for [35 S]TBPS over those consisting of β3 γ2 or β3 subunits (Slany et al., 1995; Zezula et al., 1996). GABA produces both pre- and post-synaptic inhibition in a variety of preparations by increasing the
Receptor Binding
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Picrotoxin Site of GABAA Receptors
conductance of chloride ions. One of the first compounds that was shown to block the GABA responses in crayfish was picrotoxin (Elliott and Florey, 1956; Takeuchi and Takeuchi, 1969), which was known to have a convulsant effect as early as 1875. Later studies revealed that picrotoxin antagonizes GABA responses in vertebrates in a noncompetitive manner, suggesting the possibility of different sites of action for these drugs. Picrotoxin is made up of picrotoxinin and picrotin in an equimolar ratio; picrotoxinin is 50-fold more potent than picrotin. To investigate whether there is a distinct site for picrotoxin-like convulsants at the GABA synapse, [3 H]α-dihydropicrotoxinin (DHP) was synthesized and its binding characterized (Ticku et al., 1978; Leeb-Lundberg et al., 1981). These studies revealed that the specific binding to brain membranes was only 10% to 15% of the total binding, and it was inhibited by GABAergic drugs, suggesting that the picrotoxin site at the GABA synapse is a target of drug action for several categories of centrally acting agents that affect GABAergic transmission (Ticku et al., 1978; LeebLundberg et al., 1981). Subsequently, other radioligands, such as [35 S]TBPS (or [35 S]TBPT), [3 H]TBOB, [3 H]EBOB, and [3 H]BIDN, were introduced to study the picrotoxin site of the GABAA receptor. The greatest advantage of [35 S]TBPS over [3 H]DHP is a far better signal-to-noise ratio. The binding of [35 S]TBPS to brain membranes is specific, saturable, chlorideion dependent, and modulated by GABAergic drugs (Squires et al., 1983; Ramanjaneyulu and Ticku, 1984a,b; Supavilai and Karobath, 1984; Mehta and Ticku, 1998, 1999a). Its binding to brain membranes is inhibited by picrotoxin, pentylenetetrazole, and other tetrazoles in a competitive manner (Ramanjaneyulu and Ticku, 1984a,b; Supavilai and Karobath, 1984; Mehta and Ticku, 1998, 1999a). Agonists of the benzodiazepine site enhance, whereas inverse benzodiazepine agonists inhibit [35 S]TBPS binding (Supavilai and Karobath, 1984). Muscimol, pentobarbital, and etazolate elicit biphasic effects on [35 S]TBPS binding, suggesting that these drugs interact differentially with two populations of the picrotoxin site on GABAA receptors (Supavilai and Karobath, 1984). [3 H]TBOB and [35 S]TBPS bind to the identical picrotoxin site of the GABAA receptor (Lawrence et al., 1985). However, [3 H]TBOB has a much longer radioactive half-life and binds to rat brain membranes at 0°, 24°,
and 37°C with similar Kd values, whereas [35 S]TBPS has negligible binding at 0°C. Furthermore, a shorter incubation time is required for [3 H]TBOB binding (90 versus 180 min at 24°C). In spite of these advantages, [35 S]TBPS is more commonly used as a research tool due to its lower Kd value at 24°C (20 nM versus 60 nM; Lawrence et al., 1985; Casida and Lawrence, 1985), and also because [3 H]TBOB binding is considerably less sensitive to inhibition by GABA, muscimol, and Ro 5-4864 as compared to [35 S]TBPS binding, due to differences in the nature of interactions of these radioligands with the identical picrotoxin site of the GABAA receptor (Squires et al., 1983; Ticku and Ramanjaneyulu, 1984; Lawrence et al., 1985). However, [3 H]TBOB offers the advantage of superior resolution in autoradiographic studies. [3 H]EBOB is structurally related to 3 [ H]TBOB and has high affinity (Kd 2 nM) for the picrotoxin site on the GABAA receptor and in contrast to [35 S]TBPS, also binds to invertebrate GABAA receptors (Cole and Casida, 1992; Hawkinson and Casida, 1992; Yagle et al., 2003). [3 H]BIDN is suitable for studies of GABAgated chloride channels of vertebrates as well as insects (Rauh et al., 1997). The cage convulsants (EBOB, TBPS, and TBOB) and picrotoxinin displace [3 H]BIDN binding in rat membranes but not in insect membranes, indicating that the convulsant site is somewhat different in insects (Rauh et al., 1997). However, dieldrin displaces [3 H]BIDN binding competitively in insect membranes, suggesting that the sites of action of picrotoxinin and dieldrin are not identical. Unlike picrotoxin, which antagonizes a variety of ligand-gated chloride channels, such as vertebrate glycine-gated and invertebrate L-glutamate-gated chloride channels, BIDN blocks only GABA-gated chloride channels in vertebrates and insects (Hamon et al., 1998). Kd values for [3 H]BIDN binding are 200 to 300 nM in rat brain membranes and 26 to 61 nM in insect membranes. [3 H]BIDN is not a very popular tool for studies involving rat brain membranes because of its lower affinity (higher Kd ) compared to other radioligands. Scatchard analysis and dissociation studies using various radioligands for the picrotoxin site, followed by analysis of the binding data using an iterative fitting computer program (e.g., LIGAND; Munson and Rodbard, 1980) or linear regression analysis, are utilized to investigate whether GABAergic drugs bind to the picrotoxin site or modulate this site as a result of their binding
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Table 1.18.1 IC50 and Ki Values of Various Ligands for [35 S]TBPS Binding in Rat Brain Membranesa
IC50 (mM)
Ki (µM)
Anisatin
0.10
0.09
Bemegride
200
185
6-o-Chlorophenyltetrazole
310
287
1-Cyclohexyl-5-methyl-tetrazole
40
37
6, 6 -Dichloropentamethylenetetrazole
10
9.3
Ethylbicyclophosphate
1.2
1.1
Ethyl-β–carboline-3-carboxylate (βCCE)
>2000
>2000
Heptamethylenetetrazole
230
213
1-Isobutyl-5-methylenetetrazole
480
444
Isopropylbicyclophosphate
0.16
0.15
L-Kynurenine
>1000
>1000
N-Methyl-β-carboline-carboxamide (FG7142)
>2000
>2000
1-Methyl-5-cyclohexyltetrazole
500
463
Methyl-6, 7-dimethoxy-4-ethyl-β-carboline-3 -carboxylate (DMCM)
>1000
>1000
7-Methyl-9-isopropylpentamethylenetetrazole
200
185
7-Methyl-10-isopropylpentamethylenetetrazole
145
134
7-Methylpentamethylenetetrazole
160
148
Pentylenetetrazole (PTZ)
700
648
Picrotoxinin
0.40
0.37
Quinolinic acid
120
111
Ro 5-3663
22
20
Buspirone HCl
>2000
>2000
γ-Butyrolactone
>2000
>2000
Cannabidiol
40
37
Cartazolate
0.52
0.48
Dextronantradol HCl
50
46
Diazepam
>200
>200
(±)5-(1,3-dimethylbutyl)-5-ethyl barbituric acid (DMBB)
50
46
Etazolate
5
4.6
Ethanol
500
463
(–)Etomidate
100
93
(+)Etomidate sulfate
9
8.3
Flurazepam
>200
>200
GABA
0.45
0.42
(–)Hexobarbital
380
352
(+)Hexobarbital
140
130
Ligand Convulsants
sulfate
Depressants, anticonvulsants, anxiolytics, and other drugs:
continued
Receptor Binding
1.18.15 Current Protocols in Pharmacology
Supplement 63
Table 1.18.1 IC50 and Ki Values of Various Ligands for [35 S]TBPS Binding in Rat Brain Membranesa , continued
Ligand
IC50 (mM)
Ki (µM)
(±)Hexobarbital
220
204
(–)Ketamine HCl
>2000
>2000
(+)Ketamine HCl
>2000
>2000
Levonantradol HCl
40
37
(±)Mephobarbital
210
194
Methaqualone
100
93
Metharbital
145
134
(–)N-Methyl-5-phenylpropyl barbituric acid (MPPB)
116
107
(+)N-Methyl-5-phenylpropyl barbituric acid (MPPB)
760
704
MK-801
>400
>400
(–)Pentobarbital
70
65
(+)Pentobarbital
125
116
(±)Pentobarbital
90
83
Phenobarbital
480
444
Ro 5-4864
10
9.3
Ro 11-6896
16
14.8
(–)Secobarbital
40
37
(+)Secobarbital
70
65
(±)Secobarbital
20
18.5
Tofizopam
160
148
a Drugs that inhibit specific [35 S]TBPS binding by
