Characterization of the Picrotoxin Site of GABAA Receptors

互联网2013-12-31

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes an in vitro assay for characterization of the picrotoxin site of GABA_A 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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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 [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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Figures

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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Videos

Literature Cited

Literature Cited
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Characterization of the Picrotoxin Site of GABAA Receptors

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited