CHARACTERIZATION OF HUMAN RECOMBINANT NEURONAL NICOTINIC ACETYLCHOLINE-RECEPTOR SUBUNIT COMBINATIONS ALPHA-2-BETA-4, ALPHA-3-BETA-4 AND ALPHA-4-BETA-4 STABLY EXPRESSED IN HEK293 CELLS

Human embryonic kidney (HEK293) cells were transfected with cDNA encod ing the human beta 4 neuronal nicotinic acetylcholine (ACh) receptor s ubunit in pairwise combination with human alpha 2, alpha 3 or alpha 4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha 2 and beta 4, to alph a 3 and beta 4 and to alpha 4 and beta 4 subunits, respectively. Speci fic binding of [H-3]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with K-d (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 a nd 187 +/- 29 and with B-max (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) el icit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological cha racterization of agonist-induced elevations of intracellular free Ca+ concentration revealed a distinct rank order of agonist potency for e ach subunit combination as follows: alpha 2 beta 4, (+)epibatidine (Ep i) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha 3 beta 4, Epi > DMPP = Cyt = Nic = Sub; alpha 4 beta 4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the K-b value for the compe titive antagonist dihydro-beta-erythroidine (DH beta E) was highest at alpha 3 beta 4 compared with alpha 2 beta 4 or alpha 4 beta 4 recepto rs. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell l ines are powerful tools for examining the functional and pharmacologic al properties of human alpha 2 beta 4, alpha 3 beta 4 and alpha 4 beta 4 neuronal nicotinic receptors.