title: Cell-specific effects on surface α7 nicotinic receptor expression revealed by over-expression and knockdown of rat RIC3 protein.
abstract: We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 (RIC3) by comparing RIC3 protein in rat GH4C1 and human SH-EP1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC3 exists in at least two isoforms because of an ambiguous splice site between exons 4 and 5. Both rat isoforms permit surface α7 expression in SH-EP1 and human embryonic kidney (HEK) cells measured by α-bungarotoxin binding. Contrary to expectations, endogenous RIC3 protein expression determined by immunoblots did not differ between untransfected GH4C1 or SH-EP1 cells. siRNA against rat RIC3 exon 4 and shRNA against exons 2, 5 and 6 knocked down transfected rat RIC3 expression in SH-EP1 cells and simultaneously blocked toxin binding. However, no RNAi construct blocked binding when co-transfected with α7 into GH4C1 cells. shRNA against rat exons 2 and 5 knocked down rat RIC3 protein transfected into GH4C1 cells with a time course suggesting a protein half-life of a few days. These results suggest GH4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC3 splice variants.
authors.affiliation: Department of Pharmaceutical Science, Northeastern University, Boston, MA 02115, USA.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Koperniak
authors.forename: Brijesh K
authors.initials: BK
authors.lastname: Garg
authors.forename: Jay
authors.initials: J
authors.lastname: Boltax
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Loring
mesh_terms: D017398:Alternative Splicing / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002802:Cholinesterases / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis / Q000235:genetics*; D018832:Molecular Chaperones / Q000037:antagonists & inhibitors* / Q000502:physiology*; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000235:genetics*; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: C579167:Chrna7 protein, human; C579165:Chrna7 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D018832:Molecular Chaperones; C578799:RIC3 protein, rat; D034741:RNA, Small Interfering; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D002802:Cholinesterases