Cell‐specific effects on surface α7 nicotinic receptor expression revealed by over‐expression and knockdown of rat RIC 3 protein

We tested whether surface α7 nicotinic acetylcholine receptor expression is dependent on an endogenous chaperone named Resistance to Inhibitors of Cholinesterase 3 ( RIC 3) by comparing RIC 3 protein in rat GH 4C1 and human SH ‐ EP 1 cells, which express strikingly different surface receptor levels following α7 transfection. Cloned rat RIC 3 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 ‐ EP 1 and human embryonic kidney ( HEK ) cells measured by α‐bungarotoxin binding. Contrary to expectations, endogenous RIC 3 protein expression determined by immunoblots did not differ between untransfected GH 4C1 or SH ‐ EP 1 cells. si RNA against rat RIC 3 exon 4 and sh RNA against exons 2, 5 and 6 knocked down transfected rat RIC 3 expression in SH ‐ EP 1 cells and simultaneously blocked toxin binding. However, no RNA i construct blocked binding when co‐transfected with α7 into GH 4C1 cells. sh RNA against rat exons 2 and 5 knocked down rat RIC 3 protein transfected into GH 4C1 cells with a time course suggesting a protein half‐life of a few days. These results suggest GH 4C1 cells may possess unknown chaperone(s) allowing high surface α7 expression in the absence of known RIC 3 splice variants.