Expression and functional characterisation of a human chimeric nicotinic receptor with alpha6beta4 properties.
Despite being cloned several years ago, the expression of functional nicotinic acetylcholine receptors containing the human alpha6 subunit in recombinant mammalian cell lines has yet to be demonstrated. The resulting lack of selective ligands has hindered the evaluation of the role of alpha6-containing nicotinic receptors. We report that functional channels were recorded following co-transfection of human embryonic kidney (HEK-293) cells with a chimeric alpha6/alpha4 subunit and the beta4 nicotinic receptor subunit. They displayed an agonist rank order potency of epibatidinez.>>1,1-dimethyl-4-phenylpiperazinium (DMPP)>/=cytisine>acetylcholine>nicotine measured in a fluorescent imaging plate reader assay. Nicotine, cytisine, DMPP and epibatidine displayed partial agonist properties whilst alpha-conotoxin MII and methyllycaconitine blocked the functional responses elicited by acetylcholine stimulation. Co-transfection of the alpha6/alpha4 chimera with the beta2 nicotinic receptor subunit did not result in functional receptors. The human alpha6/alpha4beta4 chimeric nicotinic receptor expressed in HEK-293 cells may provide a valuable tool for the generation of subtype specific ligands.
Evans
Non M
NM
Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor, Windlesham, GU20 6PH, Surrey, UK. Evans_Non@Lilly.com
Bose
Suchira
S
Benedetti
Giovanni
G
Zwart
Ruud
R
Pearson
Kathy H
KH
McPhie
Gordon I
GI
Craig
Peter J
PJ
Benton
Jason P
JP
Volsen
Stephen G
SG
Sher
Emanuele
E
Broad
Lisa M
LM
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D005260:Female; D015870:Gene Expression; D006801:Humans; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D014982:Xenopus laevis
D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D002118:Calcium