Rat α3/β4 Subtype of Neuronal Nicotinic Acetylcholine Receptor Stably Expressed in a Transfected Cell Line: Pharmacology of Ligand Binding and Function

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.