Characterization of the recombinant human neuronal nicotinic acetylcholine receptors α3β2 and α4β2 stably expressed in HEK293 cells

HEK293 cells were stably transfected with the cDNAs encoding full-length human 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 protein, respectively. Concentration-dependent increases in intracellular free Ca2+ concentration were elicited by nAChR agonists with a rank order of potency of EPI > 1,1-dimethyl-4-phenylpiperazinium (DMPP) > nicotine (NIC) = suberyldicholine (SUB) > cytisine (CYT) = acetylcholine (ACh) for A3B2 cells and EPI > CYT = SUB = NIC = DMPP > ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo) = mecamylamine (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 macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 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 human nAChRs are pharmacologically and functionally distinct. (C) 2000 Elsevier Science Ltd. All rights reserved.