Radioligand Binding Characterization of Neuronal Nicotinic Acetylcholine Receptors

David J. Anderson1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.8
DOI:  10.1002/0471141755.ph0108s43
Online Posting Date:  December, 2008
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Abstract

Described in this unit are radioligand binding protocols for three neuronal nicotinic acetylcholine receptor (nAChR) subtypes. Detailed descriptions of binding protocols are presented for the two predominant CNS subtypes of nAChRs, α4β2 and α7, as well as the ganglionic α3β4 nAChR. [3H]Cytisine is utilized for α4β2 nAChRs, while [3H]methyllycaconitine is utilized for α7 nAChRs, both in rat brain. α3β4 nAChRs in IMR‐32 cells are labeled with [3H]epibatidine. Reference data are presented, as well as commentary on the current state of neuronal nicotinic receptor research. Curr. Protoc. Pharmacol. 43:1.8.1‐1.8.15. © 2008 by John Wiley & Sons, Inc.

Keywords: nicotinic acetylcholine receptors; neuronal nicotinic receptors; nicotinic receptor binding; cytisine; methyllycaconitine; epibatidine; nicotine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measurement of High‐Affinity α4β2 Nicotinic Receptor Binding Sites in Brain Membrane Homogenates
  • Basic Protocol 2: Measurement of High‐Affinity α7 Nicotinic Receptor Binding Sites in Brain Membrane Homogenates
  • Basic Protocol 3: Measurement of α3β4* Nicotinic Receptor Binding Sites in IMR‐32 Cells Using [3H]Epibatidine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of High‐Affinity α4β2 Nicotinic Receptor Binding Sites in Brain Membrane Homogenates

  Materials
  • Male Sprague‐Dawley rats (250 to 400 g) or frozen tissue or membranes as described under step 1, below
  • 0.32 M sucrose, ice cold
  • Brain homogenate assay buffer 1 (see recipe), ice cold
  • Nonradiolabeled ligands (test compounds; see Table 1.8.2)
  • [3H]Cytisine (PerkinElmer Life Sciences)
  • 10 µM (−)‐nicotine
  • 0.05% polyethyleneimine (PEI; Sigma)
  • Liquid scintillation cocktail (e.g., MicroScint‐20, PerkinElmer)
    Table 1.8.2   MaterialsPharmacology of nAChR Ligands at the [3H]Cytisine, [3H]MLA, and [3H]Epibatidine (EB) Binding Sites

    Compound [3H]Cytisine binding (K i, nM) [3H]MLA binding (K i, nM) [3H]EB binding (K i, nM) b Compound source
    (±)‐Epibatidine 0.05 c 21 d 0.21 Sigma
    A‐85380 0.05 c 299 d 26 Sigma
    Varenicline e 0.08 b 144 b 99 Abbott f
    (–)‐Cytisine 0.16 g 2500 b 765 Sigma
    (+)‐Anatoxin‐A 0.31 b 122 b 36 Tocris
    (–)‐Nicotine 1.0 g 1450 d 571 Sigma
    (–)‐Lobeline 1.5 g n.d. 40 Sigma
    MCC e 1.5 g n.d. 3900 Sigma
    ABT‐418 3 j n.d. 2400 Abbott k
    DMPP e 8 g 1580 d 409 Sigma
    DHβE e 15 g >10,000 >10,000 Sigma
    GTS‐21 20 h 294 h 525 Abbott f
    Carbachol 31 g n.d. 9100 Sigma
    Methyllycaconitine >10,000 2 d 310 Sigma
    PNU 282987 e >10,000 46 d >10,000 Abbott fe,j
    α‐Bungarotoxin >10,000 17 d >10,000 Molecular Probes
    A‐582941 e >10,000 88 d 4700 Abbott fe,k
    Mecamylamine >10,000 >10,000 >10,000 Sigma

     bUnpublished data.
     cSullivan et al. ( ).
     dAnderson et al. ( ).
     eA‐582941, 2‐Methyl‐5‐(6‐phenyl‐pyridazin‐3‐yl)‐octahydro‐pyrrolo[3,4‐c]pyrrole; DHβE, dihydro‐β‐erythroidine; DMPP, 1,1‐dimethyl‐4‐phenylpiperazanium; MCC, methylcarbamylcholine; PNU‐282987, N‐[(3R)‐1‐azabicyclo[2.2.2]oct‐3‐yl]‐4‐chlorobenzamide hydrochloride, varenicline, 7,8,9,10‐tetrahydro‐6,10‐methano‐6H‐pyrazino(2,3‐h)(3)benzazepine.
     fNot commercially available.
     gAnderson and Arneric ( ).
     hBriggs et al. ( ).
     JArneric et al. ( ).
     kNow commercially available from Sigma.
     lBodnar et al. ( ).
     mTietje et al. ( ).
  • Polytron homogenizer (Brinkmann) or equivalent
  • High‐speed centrifuge (e.g., BeckmanCoulter J‐21)
  • 96‐well deep‐well polypropylene plates (optional)
  • Glass fiber filters (Whatman GF/B) or 96‐well harvest plates (Millipore or PerkinElmer)
  • Filtration apparatus (e.g., Brandel, Skatron, or PerkinElmer)
  • Liquid scintillation counter (e.g., BeckmanCoulter) or plate‐based counter (e.g., PerkinElmer Topcount)
  • Nonlinear regression curve‐fitting program, e.g., Prism (GraphPad Software)
  • Additional reagents and equipment for protein assay ( appendix 3A)

Basic Protocol 2: Measurement of High‐Affinity α7 Nicotinic Receptor Binding Sites in Brain Membrane Homogenates

  Materials
  • Brain homogenate assay buffer 2 (see recipe), ice cold
  • Brain homogenate assay buffer 2 containing 0.25% (v/v) bovine serum albumin (BSA), fraction V (Sigma)
  • [3H]Methyllycaconitine ([3H]MLA; American Radiochemical Company)
  • 2% (w/v) BSA (fraction V, Sigma)
  • Glass fiber filters (Whatman GF/B) or 96‐well harvest plates (Millipore or Perkin‐Elmer)
  • Additional reagents and equipment for measurement of α4β2 receptor binding sites ( protocol 1)

Basic Protocol 3: Measurement of α3β4* Nicotinic Receptor Binding Sites in IMR‐32 Cells Using [3H]Epibatidine

  Materials
  • IMR‐32 cell line (ATCC, CCL‐127), which expresses the human α3β4* subunit combination
  • Cell dissociation buffer: phosphate‐buffered saline (PBS) without Ca2+ or Mg2+ (e.g., Invitrogen)
  • Brain homogenate assay buffer 1 (see recipe), ice cold
  • [3H]Epibatidine (PerkinElmer Life Sciences)
  • Nonradiolabeled ligands (test compounds; see Table 1.8.2)
  • 300 nM methyllycaconitine (MLA, unlabeled) in brain homogenate assay buffer 1
  • 100 µM (−)‐nicotine
  • 0.05% polyethyleneimine (PEI)
  • Liquid scintillation cocktail (e.g., MicroScint‐20, PerkinElmer)
  • Centrifuge tubes
  • High‐speed centrifuge (e.g., BeckmanCoulter J‐21)
  • Polytron homogenizer (Brinkmann) or equivalent
  • Glass fiber filters (Whatman GF/B) or 96‐well harvest plates (Millipore or Perkin‐Elmer)
  • Filtration apparatus (e.g., Brandel, Skatron, or PerkinElmer)
  • Liquid scintillation counter (e.g., BeckmanCoulter)
  • Nonlinear regression curve‐fitting program, e.g., Prism (GraphPad Software)
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Figures

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Literature Cited

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Key References
   Anderson and Arneric, 1994. See above.
  Detailed descriptions of binding protocols for three neuronal nicotinic subtypes using rat brain homogenates and a human‐derived cell line, including characterization of reference agents in the three assay systems.
   Pabreza et al., 1991. See above.
   Houghtling et al., 1995. See above.
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