Expression of functional, recombinant ␣7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor ␣7 subunit. In addition to confirming the previously reported effect on ␣7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional ␣7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface ␣-bungarotoxin binding. Upon application of 1 mM nicotine, currents were undetectable in HEK293 cells expressing only the ␣7 subunit. In contrast, co-expression of ␣7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous ␣7 receptors. Immunoprecipitation studies demonstrate that ␣7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of ␣7 protein on the plasma membrane of cells lacking ric-3, but surface ␣-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of ␣7 receptors in HEK293 cells.
Nicotinic acetylcholine receptors (nAChRs)1 are members of the neurotransmitter-gated ion channel superfamily. They are widely expressed in the central and peripheral nervous system (1) where they influence numerous cellular and physiological processes. At least 17 different genes that code for nAChR subunits have been identified (2, 3), and they assemble as pentamers in different combinations to form a diverse set of nAChR subtypes (4, 5). The simplest case is the homopentameric complex such as that formed by the nAChR ␣7 subunit. The ␣7 receptor, for which ␣-bungarotoxin (␣-Bgt) is a specific and high affinity antagonist, is one of the most abundant receptor subtypes in the mammalian brain (6, 7). The high Ca 2ϩ permeability of the ␣7 receptor (8) suggests an involvement in the activation of Ca 2ϩ -dependent events in neurons such as transmitter release, participation in signal transduction, and a variety of modulatory effects (9). In addition, ␣7 receptors have been implicated in a number of diseases such as schizophrenia, Alzheimers, and Parkinsons disease (1, 10 -12).Heterologous expression of the ␣7 subunit in Xenopus oocytes results in homooligomeric, ␣-Bgt-sensitive receptors that activate and inactivate quickly and are highly permeable to Ca 2ϩ (8,13,14), similar to the properties of ␣7 nAChRs in neuronal cells. Although there have been reports of successful functional expression in some mammalian cell lines (15-18), measurable levels of functional receptors have been difficult to achieve in multiple cell types and this phenomenon appears to be host-cell dependent (19). The reasons for poor heterologous surface expression in these cells are not well understood. Strategies to increase the number of functional receptors on the cell...