Journal
JOURNAL OF NEUROCHEMISTRY
Volume 124, Issue 3, Pages 300-309Publisher
WILEY-BLACKWELL
DOI: 10.1111/jnc.12095
Keywords
alternate splice variants; chaperone protein; exon; receptor assembly; shRNA; siRNA
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Funding
- American Foundation for Pharmaceutical Education pre-doctoral award
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We tested whether surface a7 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 a7 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 a7 expression in SH-EP1 and human embryonic kidney (HEK) cells measured by a-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 a7 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 a7 expression in the absence of known RIC3 splice variants.
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