Characterization of the recombinant human neuronal nicotinic acetylcholinereceptors alpha 3 beta 2 and alpha 4 beta 2 stably expressed in HEK293 cells

HEK293 cells were stably transfected with the cDNAs encoding full-length hu man neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3 beta2 or alpha4 beta2. [H-3]-(+/-)Epibatidine ([H-3]-(+/-)EPI) bound to membranes from A3B2 (alpha3 beta2) and A4B2.2 (alpha4 beta2) cells with K-d values of 7.5 and 33.4 pM and B-max values of 497 and 1564 fmol/mg pro tein, respectively. Concentration-dependent increases in intracellular free Ca2+ concentration were elicited by nAChR agonists with a rank order of potency of EPI > 1,1-d imethyl-4-phenylpiperazinium (DMPP) > nicotine (NIC) = suberyldicholine (SU B) > cytisine (CYT) = acetylcholine (ACh) for A3B2 cells and EPI > CYT = SU B = NIC = DMPP > ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-in duced responses with a rank order of potency of d-tubocurarine (d-Tubo) = m ecamylamine (MEC) > dihydro-beta -erythroidine (DH betaE) in A3B2 cells and MEC = DH betaE > d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscop ic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B 2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3 beta2 and alpha4 beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca2+, respectively. Our results indicate that stably expressed alpha3 beta2 and alpha4 beta2 hu man nAChRs are pharmacologically and functionally distinct. (C) 2000 Elsevi er Science Ltd. All rights reserved.