Abstract

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human β4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human α2, α3 or α4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to α2 and β4, to α3 and β4 and to α4 and β4 subunits, respectively. Specific binding of [³H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with K_d (mean ± S.D. in pM) values of 42 ± 10, 230 ± 12 and 187 ± 29 and withB_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) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca⁺⁺concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: α2β4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; α3β4, Epi > DMPP = Cyt = Nic = Sub; α4β4, 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 competitive antagonist dihydro-β-erythroidine (DHβE) was highest at α3β4 compared with α2β4 or α4β4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human α2β4, α3β4 and α4β4 neuronal nicotinic receptors.