Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.
Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.
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1998
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通讯作者：
K. Stauderman;L. Mahaffy;M. Akong;G. Velicelebi;L. Chavez-Noriega;J. Crona;E. Johnson;K. Elliott;A. Gillespie;R. Reid;P. Adams;M. Harpold;J. Corey-Naeve
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作者：
K. Stauderman;L. Mahaffy;M. Akong;G. Velicelebi;L. Chavez-Noriega;J. Crona;E. Johnson;K. Elliott;A. Gillespie;R. Reid;P. Adams;M. Harpold;J. Corey-Naeve
用编码人β 4神经元烟碱乙酰胆碱（ACh）受体亚基与人α 2、α 3或α 4亚基成对组合的cDNA转染人胚肾（HEK 293）细胞。细胞系A2 B4、A3B4.2和A4 B4被鉴定为分别稳定表达对应于α 2和β 4、α 3和β 4以及α 4和β 4亚基的mRNA和蛋白质。在A2 B4、A3B4.2和A4 B4细胞中检测到[3 H]地棘蛙素的特异性结合，Kd（平均值+/- S.D.）以pM计）值分别为42 +/- 10、230 +/- 12和187 +/- 29，Bmax（fmol/mg蛋白质）值分别为1104 +/- 338、2010 +/- 184和3683 +/- 1450。全细胞膜片钳记录表明，（-）尼古丁（Nic）、乙酰胆碱（ACh）、野靛碱（Cyt）和碘化1，1-二甲基-4-苯基哌嗪（DMPP）均可引起瞬时内向电流。这些电流的电流-电压（I-V）关系表现出强烈的内向整流。激动剂诱导的细胞内游离Ca++浓度升高的药理学表征揭示了每个亚基组合的激动剂效力的不同等级顺序如下：α 2 β 4，（+）地棘蛙素（Epi）> Cyt >辛二酰二胆碱（Sub）= Nic = DMPP; α 3 β 4，Epi > DMPP = Cyt = Nic = Sub; α 4 β 4，Epi > Cyt = Sub > Nic > DMPP。非竞争性拮抗剂美卡拉明和d-筒箭毒碱没有显示亚型选择性。相比之下，与α 2 β 4或α 4 β 4受体相比，竞争性拮抗剂二氢-β-红霉素（DH β E）在α 3 β 4处的Kb值最高。这些数据表明，A2 B4、A3B4.2和A4 B4稳定细胞系是检查人α 2 β 4、α 3 β 4和α 4 β 4神经元烟碱受体的功能和药理学性质的有力工具。
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.