Stable expression and characterisation of a human alpha 7 nicotinic subunit chimera: a tool for functional high-throughput screening.
A chimera comprising the N-terminal region of the human alpha7 nicotinic acetylcholine receptor, fused to the transmembrane/C-terminal domains of the mouse serotonin 5-HT3 receptor, was constructed. Injection of the chimera cDNA into Xenopus oocytes, or transient transfection in human embryonic kidney (HEK-293) cells, resulted in the expression of functional channels that were sensitive to nicotinic acetylcholine, but not serotonin receptor ligands. In both systems, the responses obtained from chimeric receptors inactivated more slowly than those recorded following activation of wild-type alpha7 receptors. A stable HEK-293 cell line expressing the human alpha7/mouse 5-HT3 chimera was established, which showed that the chimera displayed a similar pharmacological profile to wild-type alpha7 receptors. Use of this chimera in high-throughput screening may enable the identification of novel pharmacological agents that will help to define further the role of alpha7 nicotinic receptors in physiology and disease.
Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor, Windlesham, Surrey GU20 6PH, UK. Peterc@lilly.com
Peter J
PJ
Craig
Suchira
S
Bose
Ruud
R
Zwart
Ruth E
RE
Beattie
Elizabeth A
EA
Folly
Laura R
LR
Johnson
Emma
E
Bell
Non M
NM
Evans
Giovanni
G
Benedetti
Kathy H
KH
Pearson
Gordon I
GI
McPhie
Stephen G
SG
Volsen
Neil S
NS
Millar
Emanuele
E
Sher
Lisa M
LM
Broad
D000109:Acetylcholine / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002678:Chimera / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics*; D012701:Serotonin / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D014982:Xenopus laevis; D064569:alpha7 Nicotinic Acetylcholine Receptor
C579167:Chrna7 protein, human; C579166:Chrna7 protein, mouse; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D012701:Serotonin; D000109:Acetylcholine