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Characterization of the Picrotoxin Site of GABAA Receptors

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Abstract
Table of Contents
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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 (K d , B max determination), association kinetics, dissociation kinetics, and competition assays (IC50 , K i determination) are included. Curr. Protoc. Pharmacol . 63:1.18.1?1.18.18. © 2013 by John Wiley & Sons, Inc.

Keywords: GABAA receptor; picrotoxin binding site; allosteric regulation; convulsants

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Introduction
Basic Protocol 1: Saturation Binding of [35S]TBPS to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes
Basic Protocol 2: Competition Assays for [35S]TBPS Binding to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes
Basic Protocol 3: Association Kinetics of Binding to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes
Basic Protocol 4: Dissociation Kinetics of Binding to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes
Alternate Protocol 1: Characterization of the Picrotoxin Site of GABAA Receptors Using [3H]TBOB
Alternate Protocol 2: Characterization of the Picrotoxin Site of GABAA Receptors Using [3H]EBOB
Alternate Protocol 3: Characterization of Picrotoxin Site of GABAA Receptor Using [3H]BIDN
Support Protocol 1: Preparation of Rat Brain Membranes
Commentary
Literature Cited
Figures
Tables

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Basic Protocol 1: Saturation Binding of [35S]TBPS to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes Materials

Frozen membrane preparation (see protocol 8Support 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, , or Prism (GraphPad)

Additional reagents and equipment for protein assays (see appendix 3A )

Basic Protocol 2: Competition Assays for [35S]TBPS Binding to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes Materials

Frozen membrane preparation (see protocol 8Support 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 )

Basic Protocol 3: Association Kinetics of Binding to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes Materials

Frozen membrane preparation (see protocol 8Support 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 )

Basic Protocol 4: Dissociation Kinetics of Binding to the Picrotoxin Site of GABAA Receptors in Rat Brain Membranes Materials

Frozen membrane preparation (see protocol 8Support 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)
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 )

Alternate Protocol 1: Characterization of the Picrotoxin Site of GABAA Receptors Using [3H]TBOB 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)

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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. K d and B max 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 () with permission from Raven Press.

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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 ( k app ) and association rate constant ( k 1 ) 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 () with permission from Raven Press.

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Literature Cited

Literature Cited
	Atack, J.R., Ohashi, Y., and McKernan, R.M. 2007. Characterization of [35S]t‐butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in postmortem human brain. Br. J. Pharmacol. 150:1066‐1074.
	Casida, J.E. and Lawrence, L.J. 1985. Structure‐activity correlations for interactions of bicyclophosphorus esters and some polychlorocycloalkane and pyrethroid insecticides with the brain‐specific t‐butylbicyclophosphorothionate receptor. Environ. Health Perspect. 61:123‐132.
	Cole, L.M. and Casida, J.E. 1992. GABA‐gated chloride channel: Binding site for 4′‐ethynyl‐4‐n‐[2,3‐3H2]propylbicycloorthobenzoate ([3H]EBOB) in vertebrate brain and insect head. Pestic. Biochem. Physiol. 44:1‐8.
	Egan, T.D., Obara, S., Jenkins, T.E., Jaw‐Tsai, S.S., Amagasu, S., Cook, D.R., Steffensen, S.C., and Beattie, D.T. 2012. AZD‐3043: A novel, metabolically labile sedative‐hypnotic agent with rapid and predictable emergence from hypnosis. Anesthesiology 116:1267‐1277.
	Elliott, K.A.C. and Florey, E. 1956. Factor I‐inhibitory factor from brain. J. Neurochem. 1:181‐182.
	Evers, A.S., Chen, Z‐W., Manion, B.D., Han, M., Jiang, X., Darbandi‐Tonkabon, R., Kable, T., Bracamontes, J., Zorumski, C.F., Mennerick, S., Steinbach, J.H., and Covey, D.F. 2010. A synthetic 18‐norsteroid distinguishes between two neuroactive steroid binding sites on GABAA receptors. J. Pharmacol. Exp. Ther. 333:404‐413.
	Hamon, A., Corronc, H.L., Hue, B., Rauh, J.J., and Sattelle, D.B. 1998. BIDN, a bicyclic dinitrile convulsant, selectively blocks GABA‐gated Cl– channels. Brain Res. 780:20‐26.
	Hawkinson, J.E. and Casida, J.E. 1992. Binding kinetics of γ‐aminobutyric acidA receptor noncompetitive antagonists: Trioxabicyclooctane, dithiane, and cyclodiene insecticide‐induced slow transition to blocked chloride channel conformation. Mol. Pharmacol. 42:1069‐1076.
	Krishnan, K., Manion, B.D., Taylor, A., Bracamontes, J., Steinbach, J.H., Reichert, D.E., Evers, A.S., Zorumski, C.F., Mennerick, S., and Covey, D.F., 2012. Neurosteroid analogues. 17. Inverted binding orientations of androsterone enantiomers at the steroid potentiation site on γ‐aminobutyric acid type A receptors. J. Med. Chem. 55:1334‐1345.
	Lawrence, L.J., Palmer, C.J., Gee, K.W., Wang, X., Yamamura, H.I., and Casida, J.E. 1985. t‐[3H]Butylbicycloorthobenzoate: New radioligand probe for the γ‐aminobutyric acid‐regulated chloride ionophore. J. Neurochem. 45:798‐804.
	Leeb‐Lundberg, F., Snowman, A., and Olsen, R.W. 1981. Perturbation of benzodiazepine receptor binding by pyrazolopyridines involves picrotoxinin/barbiturate receptor sites. J. Neurosci. 1:471‐477.
	Maksay, G. and Ticku, M.K. 1985. Dissociation of [35S]t‐butylbicyclophosphorothionate binding differentiates convulsant and depressant drugs that modulate GABAergic transmission. J. Neurochem. 44:480‐486.
	Mehta, A.K. and Ticku, M.K. 1998. Chronic ethanol administration alters the modulatory effect of 5α‐pregnan‐3α‐ol‐20‐one on the binding characteristics of various radioligands of GABAA receptors. Brain Res. 805:88‐94.
	Mehta, A.K. and Ticku, M.K. 1999a. An investigation on the role of nitric oxide in the modulation of the binding characteristics of various radioligands of GABAA receptors by 5α‐pregnan‐3α‐ol‐20‐one in the rat brain regions. Brain Res. 832:164‐167.
	Mehta, A.K. and Ticku, M.K. 1999b. An update on GABAA receptors. Brain Res. Rev. 29:196‐217.
	Munson, P.J. and Rodbard, D. 1980. LIGAND: A versatile computerized approach for characterization of ligand‐binding systems. Anal. Biochem. 107:220‐239.
	Ramanjaneyulu, R. and Ticku, M.K. 1984a. Binding characteristics and interactions of depressant drugs with [35S]t‐butylbicyclophosphorothionate, a ligand that binds to the picrotoxinin site. J. Neurochem. 42:221‐229.
	Ramanjaneyulu, R. and Ticku, M.K. 1984b. Interactions of pentamethylenetetrazole and tetrazole analogues with the picrotoxinin site of the benzodiazepine‐GABA receptor‐ionophore complex. Eur. J. Pharmacol. 98:337‐345.
	Rauh, J.J., Benner, E., Schnee, M.E., Cordova, D., Holyoke, C.W., Howard, M.H., Bai, D., Buckingham, S.D., Hutton, M.L., Hamon, A., Roush, R.T., and Sattelle, D.B. 1997. Effects of [3H]‐BIDN, a novel bicyclic dinitrile radioligand for GABA‐gated chloride channels of insects and vertebrates. Br. J.

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