Sigma Receptor Binding Assays

Uyen B. Chu1, Arnold E. Ruoho1

1 Department of Neuroscience, School of Medicine and Public Health, University of Wisconsin – Madison, Madison, WI
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.34
DOI:  10.1002/0471141755.ph0134s71
Online Posting Date:  December, 2015
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Abstract

Sigma receptors, both Sigma‐1(S1R) and Sigma‐2 (S2R), are small molecule‐regulated, primarily endoplasmic reticulum (ER) membrane‐associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)‐pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R and S2R. Methods detailed include a radioligand saturation binding assay for defining receptor densities and a competitive inhibition binding assay employing [3H]‐(+)‐pentazocine for identifying and characterizing novel ligands that interact with S1R. Procedures using [3H]‐1,3‐di(2‐tolyl)guanidine ([3H]‐DTG), a nonselective sigma receptor ligand, are described for conducting a saturation binding and competitive inhibition assays for the S2R site. These protocols are of value in drug discovery in identifying new sigma ligands and in the characterization of these receptors. © 2015 by John Wiley & Sons, Inc.

Keywords: sigma‐1 receptor; sigma‐2 receptor; saturation of radioligand binding assays; competitive inhibition of radioligand assays; [3H]‐(+)‐pentazocine; [3H]‐(+)‐DTG and haloperidol

     
 
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Table of Contents

  • Introduction
  • Sigma‐1 Receptor Radioligand‐Binding Assays
  • Basic Protocol 1: Radioligand Saturation‐Binding Assays to Determine Bmax and KD at S1R
  • Basic Protocol 2: Inhibition of [3H]‐(+)‐Pentazocine Binding to Determine Inhibition‐Binding Constants (KI) of Potential Sigma‐1 Receptor Ligands
  • Radioligand‐Binding Assays for Sigma 2 Receptors (S2R)
  • Basic Protocol 3: Radioligand Saturation‐Binding Assays to Determine KD and Bmax at S2R
  • Basic Protocol 4: Inhibition of [3H]‐1,3‐DI‐O‐Tolylguanidine Binding to Determine Inhibition Binding Constants (KI) of Potential S2R Ligands
  • Support Protocol 1: Preparation of Membrane Fractions from Tissues
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Radioligand Saturation‐Binding Assays to Determine Bmax and KD at S1R

  Materials
  • Assay buffer (see recipe)
  • 0.5 μg/μl GPL membranes (see the protocol 5Support Protocol) diluted in assay buffer (see recipe)
  • [3H]‐(+)‐pentazocine, 34 Ci/mmol (Perkin Elmer)
  • 100 μM haloperidol (Sigma‐Aldrich) dissolved in assay buffer (see recipe)
  • 0.5% Polyethyleneimine (Morris et al., ) (PEI; Sigma‐Aldrich)
  • Scintillation fluid (e.g., Ultima Gold, Perkin Elmer)
  • Scintillation counter (e.g., Packard model 1600CA, Packard Instrument)
  • 1 ml 96‐well deep‐well polypropylene plates (e.g., USA Scientifics)
  • 37°C incubator
  • Glass fiber filter papers (Whatman GF/B, Whatman)
  • Filtration system (Brandel Cell Harvester)
  • 10‐ml scintillation vials (e.g., Fisher Scientific)
  • Graphpad Prism (Graphpad Software)

Basic Protocol 2: Inhibition of [3H]‐(+)‐Pentazocine Binding to Determine Inhibition‐Binding Constants (KI) of Potential Sigma‐1 Receptor Ligands

  Materials
  • Assay buffer (see recipe)
  • 0.5 μg/μL GPL membranes (see the protocol 5Support Protocol) diluted in assay buffer (see recipe)
  • [3H]‐(+)‐pentazocine (Perkin Elmer)
  • Nonradioactive test compounds (see Table 1.34.6)
  • 100 μM haloperidol (Sigma‐Aldrich) dissolved in assay buffer (see recipe)
  • 0.5% Polyethyleneimine (Morris et al., ) (PEI; Sigma‐Aldrich)
  • Scintillation fluid (e.g., Ultima Gold, Perkin Elmer)
  • 1 ml 96‐well deep‐well polypropylene plates (e.g., USA Scientifics)
  • 37°C incubator
  • Glass fiber filter papers (Whatman GF/B, Whatman)
  • Filtration system (Brandel Cell Harvester)
  • 10‐ml scintillation vials (e.g., Fisher Scientific)
  • Scintillation counter (e.g., Packard model 1600CA, Packard Instrument)
  • Graphpad Prism (Graphpad Software)
Table 1.4.6   MaterialsSample Dilution Scheme for a Binding Site Inhibitor Tested in a Competition‐Binding Assay a

Dilution # Final unlabeled inhibitor concentration (M) 10× Stock unlabeled inhibitor concentrations (M) Dilution factor Dilution volumes
1 1.00 × 10−5 1.00 × 10−4 10 100 μl of 1.00E‐03 M stock + 900 μl of Assay Buffer
2 3.00 × 10−6 3.00× 10−5 10 100 μl of 3.00E‐04 M stock + 900 μl of Assay Buffer
3 1.00 × 10−6 1.00 × 10−5 10 100 μl of dilution #2 + 900 μl of Assay Buffer
4 3.00 × 10−7 3.00× 10−6 10 100 μl of dilution #3 + 900 μl of Assay Buffer
5 1.00 × 10−7 1.00 × 10−6 10 100 μl of dilution #4 + 900 μl of Assay Buffer
6 3.00 × 10−8 3.00 × 10−7 10 100 μl of dilution #5 + 900 μl of Assay Buffer
7 1.00 × 10−8 1.00 × 10−7 10 100 μl of dilution #6 + 900 μl of Assay Buffer
8 3.00× 10−9 3.00 × 10−8 10 100 μl of dilution #7 + 900 μl of Assay Buffer
9 1.00 × 10−9 1.00× 10−8 10 100 μl of dilution #8 + 900 μl of Assay Buffer
10 3.00 × 10−10 3.00 × 10−9 10 100 μl of dilution #9 + 900 μl of Assay Buffer

 aThe final concentration of unlabeled inhibitor is the concentration in the well (column 2). The 10× stock unlabeled inhibitor concentrations is the stock concentration prior to dilution (column 3).

Basic Protocol 3: Radioligand Saturation‐Binding Assays to Determine KD and Bmax at S2R

  Materials
  • 0.5 μg/μl GPL membranes (see the protocol 5Support Protocol) diluted in assay buffer (see recipe)
  • [3H]‐DTG, 40 Ci/mmol (Perkin Elmer)
  • 100 μM haloperidol (Sigma‐Aldrich) dissolved in assay buffer (see recipe)
  • Nonradioactive 1 μM (+)‐pentazocine (Sigma‐Aldrich) prepared in assay buffer (see recipe)
  • Assay buffer (see recipe)
  • 0.5% Polyethyleneimine (Morris et al., ) (PEI; Sigma‐Aldrich)
  • Scintillation fluid (e.g., Ultima Gold, Perkin Elmer)
  • 1‐ml 96‐well deep‐well polypropylene plates (e.g., USA Scientifics)
  • 37°C incubator
  • Glass fiber filter papers (Whatman GF/B, Whatman)
  • Filtration system (Brandel Cell Harvester)
  • 10‐ml scintillation vials (e.g., Fisher Scientific)
  • Scintillation counter (e.g., Packard model 1600CA, Packard Instrument)
  • Graphpad Prism (Graphpad Software)

Basic Protocol 4: Inhibition of [3H]‐1,3‐DI‐O‐Tolylguanidine Binding to Determine Inhibition Binding Constants (KI) of Potential S2R Ligands

  Materials
  • 0.5 μg/μl RL membranes (see the protocol 5Support Protocol) diluted in assay buffer (see recipe)
  • 0.3 μM or 300 nM [3H]‐DTG (Perkin Elmer)
  • 100 μM haloperidol (Sigma‐Aldrich) dissolved in assay buffer (see recipe)
  • 1 μM (+)‐pentazocine (Sigma‐Aldrich) dissolved in assay buffer (see recipe)
  • 0.5% Polyethyleneimine (Morris et al.) (PEI; Sigma‐Aldrich)
  • Assay buffer (see recipe)
  • Scintillation fluid (e.g., Ultima Gold, Perkin Elmer)
  • 1‐ml 96‐well deep well polypropylene plates (e.g., USA Scientifics)
  • 37°C incubator
  • Glass fiber filter papers (Whatman GF/B, Whatman)
  • Filtration system (Brandel Cell Harvester)
  • 10‐ml scintillation vials (e.g., Fisher Scientific)
  • Scintillation counter (e.g., Packard model 1600CA, Packard Instrument)
  • Graphpad Prism (Graphpad Software)

Support Protocol 1: Preparation of Membrane Fractions from Tissues

  Materials
  • Fresh or frozen liver tissue (Pel Freez Biologicals)
  • Ice
  • Homogenization buffer (see recipe)
  • Bradford Protein Assay Reagent (Thermo Scientific)
  • Liquid nitrogen
  • 50‐ml centrifuge tubes
  • Sharp dissecting scissors
  • Tissue disrupter (e.g., Brinkmann Polytron homogenizer)
  • High‐speed centrifuge or equivalent (e.g., Beckman L7 Ultracentrifuge with 45Ti rotor)
  • Tissue homogenizer (e.g., Potter‐Elvehjem Grinding Chambers and Pestles)
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Figures

Videos

Literature Cited

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