High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells

Jian Hong Peng, John D. Fryer, Raymond S. Hurst, Katherine M. Schroeder, Andrew A. George, Steven Morrissy, Vincent E. Groppi, Sherry S. Leonard, Ronald J. Lukas

Research output: Contribution to journalArticle

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Abstract

Human nicotinic acetylcholine receptor (nAChR) α7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immune-fluorescence 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 > dimethyl-phenyl-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.

Original languageEnglish (US)
Pages (from-to)24-35
Number of pages12
JournalJournal of Pharmacology and Experimental Therapeutics
Volume313
Issue number1
DOIs
StatePublished - Apr 2005
Externally publishedYes

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epibatidine
Nicotinic Receptors
Nicotine
Bungarotoxins
Binding Sites
Ligands
Tubocurarine
Protein Subunits

ASJC Scopus subject areas

  • Pharmacology

Cite this

High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells. / Peng, Jian Hong; Fryer, John D.; Hurst, Raymond S.; Schroeder, Katherine M.; George, Andrew A.; Morrissy, Steven; Groppi, Vincent E.; Leonard, Sherry S.; Lukas, Ronald J.

In: Journal of Pharmacology and Experimental Therapeutics, Vol. 313, No. 1, 04.2005, p. 24-35.

Research output: Contribution to journalArticle

Peng, Jian Hong ; Fryer, John D. ; Hurst, Raymond S. ; Schroeder, Katherine M. ; George, Andrew A. ; Morrissy, Steven ; Groppi, Vincent E. ; Leonard, Sherry S. ; Lukas, Ronald J. / High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells. In: Journal of Pharmacology and Experimental Therapeutics. 2005 ; Vol. 313, No. 1. pp. 24-35.
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abstract = "Human nicotinic acetylcholine receptor (nAChR) α7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immune-fluorescence 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 > dimethyl-phenyl-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.",
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T1 - High-affinity epibatidine binding of functional, human α7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells

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AU - Fryer, John D.

AU - Hurst, Raymond S.

AU - Schroeder, Katherine M.

AU - George, Andrew A.

AU - Morrissy, Steven

AU - Groppi, Vincent E.

AU - Leonard, Sherry S.

AU - Lukas, Ronald J.

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