Regulation by cycloheximide and lowered temperature of cell-surface alpha7-nicotinic acetylcholine receptor expression on transfected SH-EP1 cells.
Regulation by cycloheximide and lowered temperature of cell-surface alpha7-nicotinic acetylcholine receptor expression on transfected SH-EP1 cells.
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放线菌酮调节转染的 SH-EP1 细胞上细胞表面 α7-烟碱乙酰胆碱受体的表达并降低温度。
DOI：
10.1046/j.1471-4159.2003.01658.x
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发表时间：
2003
影响因子：
4.7
通讯作者：
Lukas,RonaldJ
中科院分区：
文献类型：
--
作者：
Schroeder,KatherineM;Wu,Jie;Zhao,Lingke;Lukas,RonaldJ
在哺乳动物细胞中，功能性烟碱乙酰胆碱受体（nAChR）的异源表达一直难以实现或优化，即使对于仅含有一种亚基的nAChR。在这项研究中，我们确定了降低温度或暴露于蛋白质合成抑制剂放线菌酮（CHX）对转染SH-EP 1人上皮细胞中同型α7-nAChR细胞表面表达的影响。我们发现，在25°C或存在0.5-2 μg/mL CHX的情况下孵育细胞2天，125 I标记的α-银环蛇毒素（I-Bgt）的表面结合位点分别增加了2.4倍或2.8倍。 这些增加伴随着对烟碱激动剂的峰值全细胞电流响应的增加。无论是治疗降低蛋白质合成和细胞增殖，但使用嘌呤霉素的实验表明，蛋白质合成或细胞增殖本身的减少是不足以增加表面结合。I-Bgt与全细胞膜池的结合在任一处理的反应中增加，表明表面结合的增加至少部分是由于细胞内受体水平的增加。亲环蛋白抑制剂环孢菌素A降低了未处理以及CHX或25°C处理的细胞中的表面表达。 结果表明增加细胞表面和α7-nAChR功能表达的实用方法。虽然这些作用不是简单地由于蛋白质合成抑制或细胞增殖减少，但它们确实涉及细胞内受体池大小的增加。
Heterologous expression of functional, nicotinic acetylcholine receptors (nAChR) in mammalian cells has been difficult to achieve or optimize, even for nAChR containing only one kind of subunit. In this study, we determined effects of lowered temperature or of exposure to the protein synthesis inhibitor cycloheximide (CHX) on cell surface expression of homomeric α7‐nAChR in transfected SH‐EP1 human epithelial cells. We found that incubation of cells for 2 days at 25°C or in the presence of 0.5–2 μg/mL of CHX caused ∼four‐ or ∼eight‐fold increases, respectively, in surface binding sites for125I‐labeled α‐bungarotoxin (I‐Bgt). These increases were accompanied by increases in peak whole‐cell current responses to nicotinic agonists. Either treatment lowered protein synthesis and cell proliferation, but experiments using puromycin indicated that a reduction in protein synthesis or cell proliferation per se was not sufficient to increase surface binding. I‐Bgt binding to whole‐cell membrane pools increased in response to either treatment, suggesting that the increase in surface binding was due, at least in part, to an increase in intracellular receptor levels. The cyclophilin inhibitor cyclosporin A reduced surface expression in untreated as well as CHX‐ or 25°C‐treated cells. The results suggest practical means for increasing cell surface and functional expression of α7‐nAChR. Although these effects are not simply due to protein synthesis inhibition or reduced cell proliferation, they do involve an increase in intracellular receptor pool size.