RAT ALPHA-3 BETA-4 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 ne uronal nicotinic acetylcholine receptor (nAChR) alpha 3 and beta 4 sub unit 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]e pibatidine 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 f or alpha 3/beta 4 receptor binding sites in this cell line and the bin ding sites in rat forebrain (predominantly alpha 4/beta 2 receptors) r evealed 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-erythroidin e is >7000 times higher at alpha 4/beta 2 receptors in rat forebrain t han 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 time s the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated Rb-86(+) efflux, which was blocke d by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate Rb-86(+) effluxes were 114 +/- 24 and 28 +/- 4 mu M, resp ectively. The rank order of potency of nicotinic antagonists in blocki ng the function of this alpha 3/beta 4 receptor was mecamylamine > d-t ubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompet itive mechanism, whereas dihydro-beta-erythroidine blocked the functio n competitively. The KX alpha 3 beta 4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.