title: Expression and functional characterisation of a human chimeric nicotinic receptor with alpha6beta4 properties.
abstract: 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.
authors.affiliation: Eli Lilly and Company Ltd., Lilly Research Centre, Erl Wood Manor, Windlesham, GU20 6PH, Surrey, UK. Evans_Non@Lilly.com
authors.forename: Non M
authors.initials: NM
authors.lastname: Evans
authors.forename: Suchira
authors.initials: S
authors.lastname: Bose
authors.forename: Giovanni
authors.initials: G
authors.lastname: Benedetti
authors.forename: Ruud
authors.initials: R
authors.lastname: Zwart
authors.forename: Kathy H
authors.initials: KH
authors.lastname: Pearson
authors.forename: Gordon I
authors.initials: GI
authors.lastname: McPhie
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Craig
authors.forename: Jason P
authors.initials: JP
authors.lastname: Benton
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Volsen
authors.forename: Emanuele
authors.initials: E
authors.lastname: Sher
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Broad
mesh_terms: 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
chemical_list: D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; D002118:Calcium