Characterization of Cannabinoid Receptors

Richard F. Cox1, Vincent Rash1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.26
DOI:  10.1002/0471141755.ph0126s63
Online Posting Date:  December, 2013
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This unit describes the use of cannabinoid radioligands in competitive binding assays for determining affinity parameters (IC50, Ki) of unlabeled compounds at transfected CB1 and CB2 receptors expressed in cell lines. Curr. Protoc. Pharmacol. 63:1.26.1‐1.26.10. © 2013 by John Wiley & Sons, Inc.

Keywords: cannabinoids; receptor binding; GPR 55

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • Membrane homogenates prepared from transfected cells or native tissues containing cannabinoid receptors (e.g., brain; use standard protocols to prepare membrane homogenates from cells or native tissues, see, e.g., units 1.4, 1.5, & 1.6)
  • Cannabinoid assay buffer (see recipe), room temperature and ice cold (adjust pH to 7.4 at each temperature)
  • Radioligand (e.g., [3H]rimonabant (SR141716), [3H]CP 55,940; see Table 1.26.3 and Introduction, above, for subtype selectivity; PerkinElmer NEN)
  • Unlabeled cannabinoid ligands for use as standards and to define nonspecific binding (Table 1.26.2; available, e.g., from Tocris or Sigma)
  • 100% dimethylsulfoxide (DMSO)
  • Test compounds
  • Scintillation fluid appropriate for microtiter plates (e.g., MicroScint from PerkinElmer NEN)
  • 96‐well microtiter plates (e.g., BD Falcon, cat. no. 35‐3075)
  • Opaque white and clear plate sealers
  • Platform shaker
  • 96‐well GF/C filter plates
  • 96‐well vacuum filtration membrane harvester (e.g., Packard Unifilter 196)
  • Scintillation counter capable of counting 96‐well microtiter plates (e.g., Packard TopCount)
  • Scientific graphing package (e.g., GraphPad Prism;
  • Additional reagents and equipment for radioligand binding assays (units 1.2 & 1.3)
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Literature Cited

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Key References
  Pertwee, R.G. 2010. See above.
  Contains up to date summary tables of Kd and Ki values for cannabinoid ligands.
Internet Resources
  IUPHAR Committee on Receptor Nomenclature and Drug Classification. IUPAR Database.
  A key resource on cannabinoid receptors.
  Contains a data analysis resource center covering receptor binding theory and data analysis. The online radioactivity calculator is particularly useful.
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