Rat α3/β4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line:: Pharmacology of ligand binding and function

We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha 3 and beta 4 subunit genes. This new cell line, KX alpha 3 beta 4R2, expresses a high level of the alpha 3/beta 4 receptor subtype, which binds (+/-)-[H-3]epibatidine with a K-d value of 304 +/- 16 pm and a B-max value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha 3/beta 4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha 4/beta 2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha 4/beta 2 receptors in rat forebrain than at the alpha 3/beta 4 receptors in these cells. The alpha 3/beta 4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, Rb-86(+) efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated Rb-86(+) efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate Rb-86(+) effluxes were 114 +/- 24 and 28 +/- 4 mu M, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha 3/beta 4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KX alpha 3 beta 4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.