Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel
Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel
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DOI：
10.1016/s0014-5793(02)03245-3
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发表时间：
2002-09-25
期刊：
影响因子：
3.5
通讯作者：
Lester, HA
中科院分区：
文献类型：
--
作者：
Li, P;Slimko, EM;Lester, HA
无脊椎动物的谷氨酸门控氯化物 (GluCI) 通道可被伊维菌素 (IVM) 激活，从而在哺乳动物神经元中产生电沉默。为了改进这种 GluCl/IVM 策略，我们试图突变秀丽隐杆线虫 N GluCl 通道，使其对谷氨酸不敏感，但保留对 IVM 的敏感性。基于烟碱乙酰胆碱受体超家族成员的结构功能研究，我们在卵母细胞中测试了可能参与谷氨酸反应的 β 亚基 16 个残基的 19 个点突变体。当与野生型 (WT) GluCl α 共表达时，Y182F 将谷氨酸反应降低六倍以上，而 IVM 反应几乎没有变化。对于GluClalphata(Y18217)，谷氨酸的EC50和Hill系数与WT相似，表明突变体降低了谷氨酸的功效，但不降低效力。另外，将荧光蛋白（增强型绿色荧光蛋白、增强型黄色荧光蛋白、增强型青色荧光蛋白；XFP）插入GluG α、β和β（Y182F）的M3-M4环中。我们发现这些 XFP 标记受体和 WT 受体之间没有显着的功能差异。改良的 GluCl 通道不具有谷氨酸敏感性，但带有荧光标签，可能在 GluCl 沉默策略中更有用。 (C) 2002 年欧洲生化学会联合会。由 Elsevier Science B.V. 出版。保留所有权利。
Glutamate-gated chloride (GluCI) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis eleganN GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure-function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the beta-subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six-fold, with little change to IVM responses, when coexpressed with wild-type (WT) GluCl alpha. For GluCl alphabeta(Y18217), the EC50 and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3-M4 loop of the GluG alpha, beta and beta(Y182F). We found no significant functional difference between these XFP-tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.