Stauderman K A, Mahaffy L S, Akong M, Veliçelebi G, Chavez-Noriega L E, Crona J H, Johnson E C, Elliott K J, Gillespie A, Reid R T, Adams P, Harpold M M, Corey-Naeve J
SIBIA Neurosciences, Inc., La Jolla, California 92037-4641, USA.
J Pharmacol Exp Ther. 1998 Feb;284(2):777-89.
Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (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: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 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 alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.
将编码人β4神经元烟碱型乙酰胆碱（ACh）受体亚基的cDNA与人类α2、α3或α4亚基进行两两组合，转染到人胚肾（HEK293）细胞中。鉴定出了细胞系A2B4、A3B4.2和A4B4，它们分别稳定表达与α2和β4、α3和β4以及α4和β4亚基相对应的mRNA和蛋白质。在A2B4、A3B4.2和A4B4细胞中检测到[3H]埃博霉素的特异性结合，其解离常数（Kd，以pM为单位，平均值±标准差）分别为42±10、230±12和187±29，最大结合容量（Bmax，以fmol/mg蛋白质为单位）分别为1104±338、2010±184和3683±1450。在每个细胞系中进行的全细胞膜片钳记录表明，（-）尼古丁（Nic）、ACh、金雀花碱（Cyt）和1,1 -二甲基-4 -苯基哌嗪碘化物（DMPP）可引发瞬时内向电流。这些电流的电流-电压（I-V）关系显示出强烈的内向整流。激动剂诱导细胞内游离Ca++浓度升高的药理学特性揭示了每种亚基组合激动剂效力的明显排序如下：α2β4组合中（+）埃博霉素（Epi）> Cyt >亚琥珀酰二胆碱（Sub）= Nic = DMPP；α3β4组合中Epi > DMPP = Cyt = Nic = Sub；α4β4组合中Epi > Cyt = Sub > Nic > DMPP。非竞争性拮抗剂美加明和d -筒箭毒碱没有显示出亚型选择性。相比之下，竞争性拮抗剂二氢-β-刺桐啶（DHβE）的解离常数（Kb）值在α3β4受体处最高，高于α2β4或α4β4受体处的值。这些数据表明，A2B4、A3B4.2和A4B4稳定细胞系是研究人α2β4、α3β4和α4β4神经元烟碱型受体功能和药理学特性的有力工具。
