Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals
Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals
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DOI：
10.1073/pnas.1119268109
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发表时间：
2012-04-17
影响因子：
11.1
通讯作者：
Grosman, Claudio
中科院分区：
文献类型：
--
作者：
Gonzalez-Gutierrez, Giovanni;Lukk, Tiit;Grosman, Claudio
确定离子通道各种稳定状态的结构模型是表征其构象动力学的关键步骤。在烟碱型受体的情况下，已经解决了不同的结构，但到目前为止，这些不同的模型是从超家族的不同成员中获得的。在细菌成员ELIC的情况下，来自菊花Erwinia的半胱氨酸门控通道，在没有激活配体的情况下，蛋白质的结构模型（因此，可以想象，对应于该通道的关闭状态）已经被生成。在这篇文章中，ELIC突变体的电生理特性使我们能够识别出孔隙突变，这些突变减慢了脱敏的时间过程，以至于在激动剂应用期间（bbb20分钟）通道似乎根本没有脱敏。因此，我们似乎有理由得出这样的结论：与未配体的野生型通道相比，配体结合的突变体中ELIC占据闭合状态的概率要低得多。为了深入了解ELIC在这些条件下采用的构象，我们在半胱胺浓度的存在下解决了其中两个突变体的晶体结构，该浓度引起簇内开放概率为>0.9。奇怪的是，在缺乏结合激动剂的情况下，获得的结构模型与野生型通道模型几乎无法区分。总的来说，我们的发现揭示了完整膜的功能研究在推断晶体中通道的功能状态方面的有限能力，至少在尼古丁受体超家族的情况下是如此。
The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinic-receptor superfamily.