Bridging the Gap between Structural Models of Nicotinic Receptor Superfamily Ion Channels and Their Corresponding Functional States
Bridging the Gap between Structural Models of Nicotinic Receptor Superfamily Ion Channels and Their Corresponding Functional States
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DOI：
10.1016/j.jmb.2010.09.026
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发表时间：
2010-11-12
影响因子：
5.6
通讯作者：
Grosman, Claudio
中科院分区：
文献类型：
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作者：
Gonzalez-Gutierrez, Giovanni;Grosman, Claudio
芳香族芳香族相互作用是ELIC [蛋白质数据库（PDB）代码2 VL 0]晶体结构的突出特征，ELIC是离子通道的烟碱受体超家族的细菌成员，其中五个面向孔的苯丙氨酸聚集在一起形成类似于封闭跨膜孔的窄ins的结构。我们在肌肉乙酰胆碱（ACh）受体的不同孔面位置设计了苯丙氨酸，（一次一个位置），包括与ELIC的天然苯丙氨酸246对齐的位置，并使用电生理学和毒素结合测定评估这些突变的后果。我们的结论是，面向孔的苯丙氨酸的侧链之间的相互作用，而不是其独立作用的积累，导致形成不导电的构象，该构象对ACh的应用不敏感，并且即使在没有配体的情况下也是高度稳定的。此外，来自GLIC通道的电生理记录（超家族的另一个细菌成员）被工程改造成在相应的面向孔的位置具有苯丙氨酸环，这表明这种新的不应状态与井-已知的脱敏状态似乎合理地提出，ELIC通道是在这种特殊的非导电构象中结晶的。似乎也合理地提出，在不存在面向孔的芳族侧链的环的情况下，这种稳定构象可能永远不会被ACh受体获得。我们还注意到，质子门控野生型GLIC通道对pH从7 4到4 5的快速变化的响应（在细胞外侧）仅是瞬时的，这就提出了在pH 4 0时得到的GLIC的晶体结构可能与其在室温下的晶体结构一致。（PDB代码3EHZ）和pH 4 6（PDB代码3EAM）对应于（众所周知的）脱敏状态（C）2010 Elsevier Ltd保留所有权利
Aromatic aromatic interactions are a prominent feature of the crystal structure of ELIC [Protein Data Bank (PDB) code 2VL0], a bacterial member of the nicotinic receptor superfamily of ion channels where five pore-facing phenylalanines come together to form a structure akin to a narrow ins that occludes the transmembrane pore To identify the functional state of the channel that this structure represents, we engineered phenylalanines at various pore-facing positions of the muscle acetylcholine (ACh) receptor (one position at a time), including the position that aligns with the native phenylalanine 246 of ELIC, and assessed the consequences of such mutations using electrophysiological and toxin-binding assays From our experiments, we conclude that the interaction among the side chains of pore-facing phenylalanines, rather than the accumulation of their independent effects, leads to the formation of a nonconductive conformation that is unresponsive to the application of ACh and is highly stable even in the absence of ligand Moreover, electrophysiological recordings from a GLIC channel (another bacterial member of the superfamily) engineered to have a ring of phenylalanines at the corresponding pore-facing position suggest that this novel refractory state is distinct from the well-known desensitized state It seems reasonable to propose then that it is in this peculiar nonconductive conformation that the ELIC channel was crystallized It seems also reasonable to propose that, in the absence of rings of pore-facing aromatic side chains, such stable conformation may never be attained by the ACh receptor Incidentally, we also noticed that the response of the proton-gated wild-type GLIC channel to a fast change in pH from pH 7 4 to pH 4 5 (on the extracellular side) is only transient, with the evoked current fading completely m a matter of seconds This raises the possibility that the crystal structures of GLIC obtained at pH 4 0 (PDB code 3EHZ) and pH 4 6 (PDB code 3EAM) correspond to the to the (well-known) desensitized state (C) 2010 Elsevier Ltd All rights reserved