Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.
Functional properties of human nicotinic AChRs expressed by IMR-32 neuroblastoma cells resemble those of alpha3beta4 AChRs expressed in permanently transfected HEK cells.
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DOI：
10.1085/jgp.118.5.563
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发表时间：
2001-11
期刊：
影响因子：
--
通讯作者：
Lindstrom J
中科院分区：
文献类型：
--
作者：
Nelson ME;Wang F;Kuryatov A;Choi CH;Gerzanich V;Lindstrom J
我们对人神经母细胞瘤细胞系IMR-32表达的烟碱乙酰胆碱受体（AChRs）的功能和分子特性进行了表征，并将其与稳定转染的人胚肾（HEK）细胞表达的人α3 AChRs进行了比较。IMR-32细胞与自主神经节的神经元一样，表达α3、α5、α7、β2和β4 AChR亚单位。从这些亚基中可以形成多种类型的α3 AChRs以及同源的α7 AChRs。然而，正如我们所展示的，这些细胞中功能性AChRs的性质压倒性地反映了α3β4 AChRs。α7 AChR功能未检测到，但我们估计IMR-32中的表面α7 AChR与α3 AChR相比有70%。激动剂效力（EC 50值）遵循1，1-二甲基-4-苯基哌嗪（DMPP; 16±1 μM）>尼古丁（Nic; 48 ± 7 μM）≥野靛碱（Cyt; 57 ± 3 μM）=乙酰胆碱（ACh; 59 ± 6 μM）的等级顺序。所有激动剂表现出至少80%的效力相对于乙酰胆碱。电流表现出强烈的内向整流，并以3 s−1（300 μM ACh; −60 mV）的速率脱敏。使用单克隆抗体的测定证实了IMR-32细胞中含有α3和β4的AChR占优势。α3 AChRs中含有β2亚基的比例为18%，但在细胞表面未检测到β2亚基。慢性Nic孵育增加了IMR-32细胞中的总AChR的量，但不增加表面α3β2 AChR的量。Nic孵育和降低培养温度增加了α3β2转染的HEK细胞中的总AChR和表面AChR。对HEK细胞系中表达的各种α3 AChR的表征表明，α3β4细胞系的功能特性与IMR-32细胞的功能特性最匹配。该细胞系的激动剂效力（EC 50值）的等级顺序为DMPP（14 ± 1 μM）= Cyt（18 ± 1 μM）> Nic（56 ± 15 μM）> ACh（79 ± 8 μM）。与ACh相比，Cyt和DMPP的效率均为80%左右，脱敏率为2 s-1。这些数据表明，即使具有表达几种人烟碱AChR亚型的潜力，IMR-32表达的AChR的功能特性也完全归因于α3β4 AChR。
We characterized the functional and molecular properties of nicotinic acetylcholine receptors (AChRs) expressed by IMR-32, a human neuroblastoma cell line, and compared them to human α3 AChRs expressed in stably transfected human embryonic kidney (HEK) cells. IMR-32 cells, like neurons of autonomic ganglia, have been shown to express α3, α5, α7, β2, and β4 AChR subunits. From these subunits, several types of α3 AChRs as well as homomeric α7 AChRs could be formed. However, as we show, the properties of functional AChRs in these cells overwhelmingly reflect α3β4 AChRs. α7 AChR function was not detected, yet we estimate that there are 70% as many surface α7 AChRs in IMR-32 when compared with α3 AChRs. Agonist potencies (EC50 values) followed the rank order of 1,1-dimethyl-4-phenylpiperazinium (DMPP; 16±1 μM) > nicotine (Nic; 48 ± 7 μM) ≥ cytisine (Cyt; 57 ± 3 μM) = acetylcholine (ACh; 59 ± 6 μM). All agonists exhibited efficacies of at least 80% relative to ACh. The currents showed strong inward rectification and desensitized at a rate of 3 s−1 (300 μM ACh; −60 mV). Assays that used mAbs confirmed the predominance of α3- and β4-containing AChRs in IMR-32 cells. Although 18% of total α3 AChRs contained β2 subunits, no β2 subunit was detected on the cell surface. Chronic Nic incubation increased the amount of total, but not surface α3β2 AChRs in IMR-32 cells. Nic incubation and reduced culture temperature increased total and surface AChRs in α3β2 transfected HEK cells. Characterization of various α3 AChRs expressed in HEK cell lines revealed that the functional properties of the α3β4 cell line best matched those found for IMR-32 cells. The rank order of agonist potencies (EC50 values) for this line was DMPP (14 ± 1 μM) = Cyt (18 ± 1 μM) > Nic (56 ± 15 μM > ACh (79 ± 8 μM). The efficacies of both Cyt and DMPP were ∼80% when compared with ACh and the desensitization rate was 2 s−1. These data show that even with the potential to express several human nicotinic AChR subtypes, the functional properties of AChRs expressed by IMR-32 are completely attributable to α3β4 AChRs.
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