Recent studies have shown that RIC-3, originally identified in C. elegans as the protein encoded by the gene
ric-3 (resistance to inhibitors of cholinesterase), can enhance functional expression of alpha7 nicotinic acetylcholine receptors (nAChRs). In the present study, the influence of RIC-3 upon multiple homomeric (alpha7, alpha8 and alpha9) and heteromeric (alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4 and alpha9alpha10) nAChR subtypes has been examined in transfected mammalian cells by radioligand binding and functional characterization. Coexpression of RIC-3 facilitates a dramatic enhancement of the ability of alpha7 (and the closely related alpha8 subunit) to generate functional nAChRs in otherwise non-permissive mammalian cells. In contrast, coexpression of RIC-3 did not facilitate functional expression of either homomeric alpha9 or heteromeric alpha9alpha10 nAChRs in mammalian cell lines. Interestingly, whereas RIC-3 has been reported to cause a marked functional inhibition of heteromeric nAChRs such as alpha3beta4 and alpha4beta2 expressed in Xenopus oocytes, RIC-3 significantly enhances levels of functional expression of these and other (alpha3beta2 and alpha4beta4) heteromeric nAChRs when expressed in mammalian cell lines. In addition, the interaction of multiple nAChR subunits (alpha3, alpha4, alpha7, beta2 and beta4) with RIC-3 has been demonstrated by coimmunoprecipitation from metabolically labelled transfected cells. Significantly, coimmunoprecipitation experiments have provided evidence that RIC-3 associates with unassembled nAChR subunits, a finding which is consistent with previous suggestions that RIC-3 may act by enhancing the maturation (subunit folding and assembly) of nAChRs. We conclude that RIC-3 is a nAChR-associated protein which can enhance functional expression of multiple nAChR subtypes in transfected mammalian cells.