High-Affinity Epibatidine Binding of Functional, Human α7-Nicotinic Acetylcholine Receptors Stably and Heterologously Expressed de Novo in Human SH-EP1 Cells

  1. Jian-Hong Peng,
  2. John D. Fryer1,
  3. Raymond S. Hurst2,
  4. Katherine M. Schroeder3,
  5. Andrew A. George4,
  6. Steven Morrissy5,
  7. Vincent E. Groppi6,
  8. Sherry S. Leonard and
  9. Ronald J. Lukas
  1. Division of Neurobiology, Barrow Neurological Institute, Phoenix, Arizona (J.-H.P., J.D.F., K.M.S., A.A.G., S.M., R.J.L.); Pharmacia Corporation, Kalamazoo, Michigan (R.S.H., V.E.G.); and Department of Psychiatry, University of Colorado Health Sciences Center, Denver, Colorado (S.S.L.)
  1. Address correspondence to:
    Dr. R. J. Lukas, Division of Neurobiology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013. E-mail: rlukas{at}chw.edu

Abstract

Human nicotinic acetylcholine receptor (nAChR) α7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows α7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled α-bungarotoxin (I-Bgt) binding sites corresponding to human α7 (hα7)-nAChRs on the surface of most cells. I-Bgt binds to hα7-nAChRs in membrane fractions with a typical apparent KD value of ∼5 nM and Bmax value of ∼1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed hα7-nAChRs is evident as rapid, transient inward current responses to (–)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to ∼100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing α3 or α4 subunits (KD values of about 100 or 10 pM, respectively). hα7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (KD value of ∼0.6 nM). Studies of several forms of native or heterologously expressed rat or human α7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and α-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine ∼ cytisine > carbamylcholine ∼ d-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human α7-nAChRs that exhibit ligand binding and functional properties like native α7-nAChRs and that epibatdine is useful as a probe for human α7-nAChRs.

Footnotes

  • The work done in Phoenix was supported by endowment and capitalization funds from the Men's and Women's Boards of the Barrow Neurological Foundation and Epi-Hab Phoenix, Inc., by the Arizona Disease Control Research Commission (Grants 9730 and 9615), the National Institutes of Health (Grant NS40417), and the Council for Tobacco Research–U.S.A., Inc. (Grant 4366). Work done in Denver was supported by the Veterans Affairs Medical Research Service and National Institutes of Health Grants DA9457 and DA12241. The contents of this report are solely the responsibility of the authors and do not necessarily represent the views of the aforementioned awarding agencies. This article is dedicated to the memory of Jian-Hong Peng and Michelle Zhang. Portions of this work were presented previously [Peng J-H, Leonard SS, and Lukas RJ (1998) Heterologous expression of epibatidine- and α-bungarotoxin-binding human α7-nicotinic acetylcholine receptor in a native receptor-null human epithelial cell line. Soc Neurosci Abstr24:831].

  • doi:10.1124/jpet.104.079004.

  • ABBREVIATIONS: nAChR, nicotinic acetylcholine receptor; α3*-nAChR or α4*-nAChR, accepted nomenclature for nAChR containing α3 or α4 subunits, respectively, as well as other nAChR subunits; Bgt, α-bungarotoxin; hα7, human α7 subunit(s); hα7-nAChR, human α7-nicotinic acetylcholine receptor(s); I-Bgt, 125I-labeled α-bungarotoxin; PBS, phosphate-buffered saline; MLA, methyllycaconitine; DMPP, 1,1-dimethyl-4-phenyl-piperazinium; EBDN, epibatidine.

  • ↵1 Current address: Division of Biology and Medical Sciences, Washington University, St. Louis, MO.

  • ↵2 Current address: Pfizer, Groton, CT.

  • ↵3 Current address: Phoenix Epidemiology and Clinical Research Branch, National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, Phoenix, AZ.

  • ↵4 Current address: Institute for Neuroscience, University of Texas at Austin, Austin, TX.

  • ↵5 Current address: College of Medicine, University of Arizona, Tucson, AZ.

  • ↵6 Current address: Pfizer, Kalamazoo, MI.

    • Received October 7, 2004.
    • Accepted December 6, 2004.
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  1. Published online before print December 8, 2004, doi: 10.1124/jpet.104.079004 JPET April 2005 vol. 313 no. 1 24-35
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