RIC-3 Enhances Functional Expression of Multiple Nicotinic Acetylcholine Receptor Subtypes in Mammalian Cells

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Abstract
Recent studies have shown that RIC-3, originally identified in Caenorhabditis elegans as the protein encoded by the gene resistance to inhibitors of cholinesterase (ric-3), can enhance functional expression of α7 nicotinic acetylcholine receptors (nAChRs). In the present study, the influence of C. elegans and human RIC-3 upon multiple homomeric (α7, α8, and α9) and heteromeric (α3β2, α3β4, α4β2, α4β4, and α9α10) 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 α7 (and the closely related α8 subunit) to generate functional nAChRs in otherwise nonpermissive mammalian cells. In contrast, coexpression of RIC-3 did not facilitate functional expression of either homomeric α9 or heteromeric α9α10 nAChRs in mammalian cell lines. It is noteworthy that whereas RIC-3 has been reported to cause a marked functional inhibition of heteromeric nAChRs such as α3β4 and α4β2 expressed in Xenopus laevis oocytes, RIC-3 significantly enhances levels of functional expression of these and other (α3β2 and α4β4) heteromeric nAChRs when expressed in mammalian cell lines. In addition, the interaction of multiple nAChR subunits (α3, α4, α7, β2, and β4) with RIC-3 has been demonstrated by coimmunoprecipitation from metabolically labeled transfected cells. It is significant that coimmunoprecipitation experiments have provided evidence that RIC-3 associates with unassembled nAChR subunits, a finding that 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 an nAChR-associated protein that can enhance functional expression of multiple nAChR subtypes in transfected mammalian cells.