In Vitro Muscarinic Receptor Radioligand‐Binding Assays

Tod Steinfeld1, M. Teresa Pulido‐Rios1, Scott Armstrong1, Sharath S. Hegde1

1 Theravance, South San Francisco, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.33
DOI:  10.1002/0471141755.ph0133s48
Online Posting Date:  March, 2010
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Abstract

G‐protein‐coupled muscarinic receptors (mAChRs), of which there are five subtypes (M1‐M5), are attractive drug targets for a number of disorders. Described in this unit are radioligand‐binding assays for defining the selectivity and affinity of chemical agents at the five mAChR subtypes. Detailed methodologies and troubleshooting strategies are provided for saturation‐binding studies, to estimate KD and Bmax values, and for competition‐binding studies to estimate Ki values. Emphasis is placed on experimental details that are critical for executing a robust and reliable assay. Curr. Protoc. Pharmacol. 48:1.33.1‐1.33.18. © 2010 by John Wiley & Sons, Inc.

Keywords: muscarinic; acetylcholine; radioligand; binding

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Radioligand Saturation‐Binding Assays to Determine Bmax and KD of Ligands at Muscarinic Receptors
  • Basic Protocol 2: Determination of Inhibition Binding Constants (KI) by Radioligand Binding
  • Support Protocol 1: Preparation of Membrane Fractions from Tissues
  • Support Protocol 2: Preparation of Membrane Fractions from Cultured Cells
  • Support Protocol 3: Determination of Radioligand Concentration
  • Support Protocol 4: Analysis of Competition Binding Experiments
  • 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 of Ligands at Muscarinic Receptors

  Materials
  • Membrane preparation from organ tissues or cell cultures (see Support Protocols protocol 31 and protocol 42)
  • Assay buffer (see recipe)
  • Protein quantification assay kit (e.g., Bio‐Rad BCA reagent and protein standards)
  • [3H]NMS (e.g., Perkin Elmer, cat. no. NET63001MC)
  • Scintillation cocktail (e.g., Perkin‐Elmer Microscint‐20 and Beckman Ready Safe)
  • Dilution buffer (see recipe)
  • 10 mM atropine (see recipe)
  • 0.3% polyethyleneimine (PEI; see recipe)
  • Wash buffer (see recipe), ice‐cold
  • Tissue disrupter (e.g., Kinematics Polytron PT‐2100)
  • Clear polystyrene plate (e.g., Corning RIA plate)
  • Spectrophotometric microplate reader (e.g., Molecular Devices SpectraMax)
  • Polypropylene 96‐well deepwell blocks (e.g., Corning Assay Block)
  • 5‐ml scintillation vials (e.g., Beckman Poly‐Q vials)
  • Scintillation counters (e.g., Beckman LS‐6500 and Perkin‐Elmer TopCount)
  • GF/B filter paper or plates (e.g., Perkin‐Elmer Unifilter plates)
  • Filtration system (e.g., Brandel Cell Harvester or Perkin‐Elmer FilterMate)
  • Additional reagents and equipment for measuring protein concentration ( appendix 3A)

Basic Protocol 2: Determination of Inhibition Binding Constants (KI) by Radioligand Binding

  Materials
  • Membrane preparation (see Support Protocols protocol 31 and protocol 42)
  • Assay buffer (see recipe)
  • Protein assay kit (Bio‐Rad BCA reagent and protein standards)
  • Inhibitors (see Table 1.33.7 for suggested compounds)
  • [3H]NMS (see protocol 5)
  • Scintillation cocktail (e.g., Perkin‐Elmer Microscint‐20 and Beckman Ready Safe)
  • Dilution buffer (see recipe)
  • 10 mM atropine (see recipe)
  • 0.3% polyethyleneimine (PEI; see recipe)
  • Wash buffer (see recipe), ice‐cold
  • Tissue disrupter (e.g., Kinematics Polytron PT‐2100)
  • Polystyrene RIA plate (e.g., Corning)
  • Spectrophotometric microplate reader (e.g., Molecular Devices SpectraMax)
  • Polypropylene 96‐well deepwell blocks (e.g., Costar Assay Block)
  • 5‐ml scintillation vials (e.g., Beckman poly‐Q vials)
  • Scintillation counters (e.g., Beckman LS‐6500 and Perkin‐Elmer TopCount)
  • GF/B filter paper or plates (e.g., Perkin‐Elmer Unifilter plates)
  • Filtration system (e.g., Brandel Cell Harvester or Perkin‐Elmer FilterMate)
  • Additional reagents and equipment for measuring protein concentration ( appendix 3A)

Support Protocol 1: Preparation of Membrane Fractions from Tissues

  Materials
  • Fresh or frozen tissue expressing muscarinic receptors such as heart, brain, lung, bladder, or submaxillary gland
  • Lysis buffer A (see recipe)
  • Assay buffer (see recipe)
  • Tissue disrupter (e.g., Kinematica Polytron PT‐2100 with PT DA 2112 generator)
  • Benchtop centrifuge (e.g., Beckman Allegra 6R with GH‐3.8 rotor and 50‐ml tube swinging buckets)
  • 50‐ml conical centrifuge tubes (e.g., Becton Dickinson 50‐ml Falcon tubes)
  • Cheesecloth
  • High‐speed centrifuge (e.g., Beckman J2‐MC with JA‐17 rotor)
  • Polycarbonate centrifuge tubes (e.g., Oakridge 30‐ml tubes)

Support Protocol 2: Preparation of Membrane Fractions from Cultured Cells

  Materials
  • Cultured cells expressing muscarinic receptors (e.g., the authors used CHO‐K1 cells stably transfected with human recombinant M 1‐M 4)
  • Lysis buffer B (see recipe)
  • Assay buffer (see recipe)
  • Tissue disrupter (e.g., Kinematica Polytron PT‐2100 with PT DA 2112 generator)
  • Conical centrifuge tubes (e.g., Becton Dickinson 50‐ml Falcon tubes)
  • Benchtop centrifuge (e.g., Beckman Allegra 6R with GH‐3.8 rotor and 50‐ml tube swinging buckets)
  • Polycarbonate centrifuge tubes (e.g., Oakridge 30‐ml tubes)
  • High‐speed centrifuge (e.g., Beckman J2‐MC with JA‐17 rotor)
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Figures

Videos

Literature Cited

Literature Cited
   Caulfield, M.P. and Birdsall, N.J.M. 1998. International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev. 50:279‐290.
   Cheng, Y. and Prusoff, W.H. 1973. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099‐3108.
   Ellis, J., Huyler, J., and Brann, M.R. 1991. Allosteric regulation of cloned M1‐M5 muscarinic receptor subtypes. Biochem. Pharmacol. 42:1927‐1932.
   Jolkkonen, M., van Giersbergen, P.L.M., Hellman, U., Wernstedt, C., and Karlsson, E. 1994. A toxin from the green mamba Dendroaspis angusticeps: Amino acid sequence and selectivity for muscarinic M4 receptors. FEBS Lett. 352:91‐94.
   Kenakin, T. 2006. A Pharmacology Primer: Theory, Application and Methods, 2nd ed. Academic Press, San Diego.
   Motulsky, H.J. and Mahan, L.C. 1984. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25:1‐9.
   Rosenthal, H.E. 1967. A graphic method for the determination and presentation of binding parameters in a complex system. Anal. Biochem. 20:525‐532.
   Wess, J., Eglen, R.M., and Gautam, D. 2007. Muscarinic acetylcholine receptors: Mutant mice provide new insights for drug development. Nat. Rev. Drug Discov. 6:721‐733.
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