Characterization of the Picrotoxin Site of GABAA Receptors

Ashok K. Mehta1, Maharaj K. Ticku1

1 The University of Texas Health Science Center, San Antonio, Texas
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.18
DOI:  10.1002/0471141755.ph0118s63
Online Posting Date:  December, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

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. Pharmacol. 63:1.18.1‐1.18.18. © 2013 by John Wiley & Sons, Inc.

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

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • 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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

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 [35S]t‐butylbicyclophosphorothionate ([35S]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 [35S]t‐butylbicyclophosphorothionate ([35S]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 [35S]t‐butylbicyclophosphorothionate ([35S]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 [35S]t‐butylbicyclophosphorothionate ([35S]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)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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. Pharmacol. 121:1496‐1505.
  Rey, M., Kruse, M.S., Alvarez, L.D., Ghini, A.A., Veleiro, A.S., Burton, G., and Coirini, H. 2013. Neuroprotective action of synthetic steroids with oxygen bridge. Activity on GABAA receptor. Exp. Neurol. Epub ahead of print doi:10.1016/j.expneurol.2013.07.020.
  Slany, A., Zezula, J., Tretter, V., and Sieghart, W. 1995. Rat β3 subunits expressed in human embryonic kidney 293 cells form high affinity [35S]t‐butylbicyclophosphorothionate binding sites modulated by several allosteric ligands of γ‐aminobutyric acid type A receptors. Mol. Pharmacol. 48:385‐391.
  Squires, R.F., Casida, J.E., Richardson, M., and Saederup, E. 1983. [35S]t‐Butylbicyclophosphorothionate binds with high affinity to brain specific sites coupled to γ‐aminobutyric acidA and ion recognition sites. Mol. Pharmacol. 23:326‐336.
  Supavilai, P. and Karobath, M. 1984. [35S]t‐Butylbicyclophosphorothionate binding sites are constituents of the γ‐aminobutyric acid‐benzodiazepine receptor complex. J. Neurosci. 4:1193‐1200.
  Takeuchi, A. and Takeuchi, T. 1969. A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish. J. Physiol. 205:377‐391.
  Ticku, M.K. and Ramanjaneyulu, R. 1984. Ro 5‐4864 inhibits the binding of [35S]t‐butylbicyclophosphorothionate to rat brain membranes. Life Sci. 34:631‐638.
  Ticku, M.K., Ban, M., and Olsen, R.W. 1978. Binding of [3H]α‐dihydropicrotoxinin, a γ‐aminobutyric acid synaptic antagonist, to rat brain membranes. Mol. Pharmacol. 14:391‐402.
  Yagle, M.A., Martin, M.W., de Fiebre, C.M., de Fiebre, N.C., Drewe, J.A. and Dillon, G.H. 2003. [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors. Neurotoxicology 24:817‐824.
  Zezula, J., Slany, A., and Sieghart, W. 1996. Interaction of allosteric ligands with GABAA receptors containing one, two, or three different subunits. Eur. J. Pharmacol. 301:207‐214.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library