Abstract

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) α3 and β4 subunit genes. This new cell line, KXα3β4R2, expresses a high level of the α3/β4 receptor subtype, which binds (±)- [³H]epibatidine with aK _d value of 304±16 pm and a B _max value of 8942 ± 115 fmol/mg protein. Comparison of nicotinic drugs in competing for α3/β4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly α4/β2 receptors) revealed marked differences in theirK _i values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-β-erythroidine is >7000 times higher at α4/β2 receptors in rat forebrain than at the α3/β4 receptors in these cells. The α3/β4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, ⁸⁶Rb⁺ efflux was increased to levels 8–10 times the basal levels. Acetylcholine, (−)-nicotine, cytisine, carbachol, and (±)-epibatidine all stimulated⁸⁶Rb⁺ efflux, which was blocked by mecamylamine. The EC₅₀ values for acetylcholine and (−)-nicotine to stimulate ⁸⁶Rb⁺ effluxes were 114 ± 24 and 28 ± 4 μm, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this α3/β4 receptor was mecamylamine >d-tubocurarine > dihydro-β-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-β-erythroidine blocked the function competitively. The KXα3β4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.