title: Ric-3 promotes functional expression of the nicotinic acetylcholine receptor alpha7 subunit in mammalian cells.
abstract: Expression of functional, recombinant alpha7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of alpha7 receptors in HEK293 cells.
authors.affiliation: Merck Research Laboratories, San Diego, California 92121, USA.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Williams
authors.forename: Bill
authors.initials: B
authors.lastname: Burton
authors.forename: Arturo
authors.initials: A
authors.lastname: Urrutia
authors.forename: Anatoly
authors.initials: A
authors.lastname: Shcherbatko
authors.forename: Laura E
authors.initials: LE
authors.lastname: Chavez-Noriega
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Cohen
authors.forename: Jayashree
authors.initials: J
authors.lastname: Aiyar
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002038:Bungarotoxins / Q000378:metabolism; D002460:Cell Line; D004553:Electric Conductivity; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins; D009474:Neurons / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D002038:Bungarotoxins; C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D021122:Protein Subunits; D011506:Proteins; C496701:RIC3 protein, human; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor