Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions.
Site-directed spin labeling reveals pentameric ligand-gated ion channel gating motions.
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DOI：
10.1371/journal.pbio.1001714
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发表时间：
2013-11
期刊：
影响因子：
9.8
通讯作者：
Czajkowski C
中科院分区：
文献类型：
--
作者：
Dellisanti CD;Ghosh B;Hanson SM;Raspanti JM;Grant VA;Diarra GM;Schuh AM;Satyshur K;Klug CS;Czajkowski C
五聚体配体门控离子通道(PLGICs)是神经递质激活的受体，介导快速突触传递。在pLGICs中，激动剂与胞外区的结合触发结构重排，导致跨膜区离子传导孔的打开，在神经递质的持续存在下，通道脱敏(关闭)。连接细胞外结合结构域(环2、7和9)和跨膜通道结构域(M2-M3环)的每个亚基中的柔性环对于将配体结合耦合到通道门控是必不可少的。比较两种细菌pLGIC同源物的晶体结构，ELIC和质子激活的GLIC表明通道门控与这些环中的重排有关，但这些运动是否准确地预测了功能性脂质嵌入的pLGIC中的运动尚不清楚。在这里，利用定点定向自旋标记(SDSL)电子顺磁共振(EPR)谱和重组成脂质体的功能性GLIC通道，我们研究了在质子依赖的门控从静止状态到不敏感状态的转变过程中，ECD/TMD门控界面的环路是否以及移动了多远。环路9响应质子诱导的脱敏反应，将∼9？向内移向通道管腔。当GLIC处于洗涤剂胶束中时，没有观察到环路9的运动，这表明洗涤剂的增溶作用捕获了处于非激活状态的蛋白质，而脂质是功能门控转换所必需的。质子诱导的脱敏作用使环2固定，位置变化很小。没有观察到M2-M3环的质子诱导运动，这表明它在闭合和钝化状态下的构象几乎相同。我们的自旋标记GLIC的实验距离测量表明，ELIC不是一个很好的GLIC功能休眠状态的模型，并且GLIC的晶体结构并不对应于钝化状态。这些发现促进了我们对pLGIC门控的分子机制的理解。配体门控离子通道存在于神经和肌肉细胞的膜上。这些蛋白质形成跨越膜的通道，在那里它们将化学信号转化为电兴奋性的变化。神经递质与这些蛋白质的细胞外表面结合，触发全球结构重排，打开通道，允许离子流过细胞膜。在神经递质持续存在的情况下，这些通道会脱敏并关闭。通道的开启和关闭调节大脑中的肌肉收缩和信号传递，而这些通道的缺陷会导致各种疾病。虽然晶体结构提供了这些蛋白质在假定的关闭和开放通道状态下的冰冻快照，但人们对这些通道在实际信号事件中如何脱敏和移动知之甚少。在这里，我们应用了一种技术来研究蛋白质的结构和局部动力学，这种技术被称为定位自旋标记到一个典型的配体门控通道GLIC。我们直接量化了配体诱导的运动在结合结构域(环2和9)和通道结构域(M2-M3环)之间的区域。我们发现，环9的大幅移动和环2的固定重新排列了结合结构域和通道结构域之间的界面，伴随着GLIC通道门控转变到不敏感状态。这些数据为蛋白质运动提供了新的见解，蛋白质运动是细胞间信号电化学传输的基础。
Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, binding of agonist to the extracellular domain triggers a structural rearrangement that leads to the opening of an ion-conducting pore in the transmembrane domain and, in the continued presence of neurotransmitter, the channels desensitize (close). The flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, and 9) to the transmembrane channel domain (M2–M3 loop) are essential for coupling ligand binding to channel gating. Comparing the crystal structures of two bacterial pLGIC homologues, ELIC and the proton-activated GLIC, suggests channel gating is associated with rearrangements in these loops, but whether these motions accurately predict the motions in functional lipid-embedded pLGICs is unknown. Here, using site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and functional GLIC channels reconstituted into liposomes, we examined if, and how far, the loops at the ECD/TMD gating interface move during proton-dependent gating transitions from the resting to desensitized state. Loop 9 moves ∼9 Å inward toward the channel lumen in response to proton-induced desensitization. Loop 9 motions were not observed when GLIC was in detergent micelles, suggesting detergent solubilization traps the protein in a nonactivatable state and lipids are required for functional gating transitions. Proton-induced desensitization immobilizes loop 2 with little change in position. Proton-induced motion of the M2–M3 loop was not observed, suggesting its conformation is nearly identical in closed and desensitized states. Our experimentally derived distance measurements of spin-labeled GLIC suggest ELIC is not a good model for the functional resting state of GLIC, and that the crystal structure of GLIC does not correspond to a desensitized state. These findings advance our understanding of the molecular mechanisms underlying pLGIC gating. Ligand-gated ion channels reside in the membranes of nerve and muscle cells. These proteins form channels that span the membrane, where they transduce chemical signals into changes in electrical excitability. Neurotransmitters bind to the extracellular surface of these proteins to trigger global structural rearrangements that open the channel, allowing ions to flow across the cell membrane. In the continued presence of neurotransmitters, the channels desensitize and close. Channel opening and closing regulate muscle contraction and signaling in the brain, and defects in these channels lead to a variety of diseases. While crystal structures have provided frozen snapshots of these proteins in presumed closed and open channel states, little is known about how the channels desensitize and move during actual signaling events. Here, we applied a technique to investigate the structure and local dynamics of proteins known as site-directed spin labeling to a prototypical ligand-gated channel, GLIC. We directly quantified ligand-induced motions in regions at the boundary between the binding domain (loops 2 and 9) and the channel domain (M2–M3 loop). We show that a large movement of loop 9 and an immobilization of loop 2, which rearranges the interface between the binding and channel domains, accompanies GLIC channel gating transitions into a desensitized state. These data provide new insights into the protein movements that underlie electrochemical transmission of signals between cells.