Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor.
The alpha 7 neuronal nicotinic acetylcholine receptor subtype forms a Ca(2+)-permeable homooligomeric ion channel sensitive to alpha-bungarotoxin in Xenopus oocytes. In this study, we have stably and functionally expressed the human alpha 7 cDNA in a mammalian cell line, HEK-293 and examined its pharmacologic properties. [125I] alpha-Bungarotoxin bound to transfected cells with a Kd value of 0.7 nM and a Bmax value of 973 pmoL/mg protein. No specific binding was detected in untransfected cells. Specific binding could be displaced by unlabeled alpha-bungarotoxin (Ki = 0.5 nM) and an excellent correlation was observed between binding affinities of a series of nicotinic cholinergic ligands in transfected cells and those in the human neuroblastoma IMR-32 cell line. Additionally, cell surface expression of alpha 7 receptors was detected by fluorescein isothiocyanate-conjugated alpha-bungarotoxin in transfected cells. Whole cell currents sensitive to blockade by alpha-bungarotoxin, and with fast kinetics of activation and inactivation, were recorded from transfected cells upon rapid application of (-)-nicotine or acetylcholine with EC50 values of 49 microM and 155 microM respectively. We conclude that the human alpha 7 subunit when expressed alone can form functional ion channels and that the stably transfected HEK-293 cell line serves as a unique system for studying human alpha 7 nicotinic receptor function and regulation, and for examining ligand interactions.
Gopalakrishnan
M
M
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064-3500, USA.
Buisson
B
B
Touma
E
E
Giordano
T
T
Campbell
J E
JE
Hu
I C
IC
Donnelly-Roberts
D
D
Arneric
S P
SP
Bertrand
D
D
Sullivan
J P
JP
D000109:Acetylcholine / Q000378:metabolism; D001483:Base Sequence; D002038:Bungarotoxins / Q000494:pharmacology; D002460:Cell Line; D004594:Electrophysiology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009538:Nicotine / Q000378:metabolism; D018733:Nicotinic Antagonists / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D012313:RNA / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000187:drug effects*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D002038:Bungarotoxins; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D012313:RNA; D009538:Nicotine; D000109:Acetylcholine