LARGE SCALE MD SIMULATIONS OF ANESTHETIC EFFECTS ON ION CHANNELS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们研究的最终目的是了解全身麻醉的分子机制。我们的计算工作的直接目标是引出蛋白质运动的分子细节，这些分子细节很难用实验方法直接观察，但对理解通道蛋白的麻醉作用可能至关重要。我们将重点关注被认为是全身麻醉药的潜在靶点的nAChR。研究的具体目标包括：（a）进行大规模MD模拟（根据需要10 ns或更长，3次重复，使用不同的种子）在不存在麻醉剂的情况下，在3：1：1 POPC：POPA：胆固醇的完全水合膜贴片中，对（<$1）2 <$1 <$$>和神经元（<$4）2（<$2）3和（<$7）5 nAChR的开放通道结构的影响（控制系统）;（B）识别麻醉剂（氟烷和异氟烷）相互作用位点在鱼雷（<$1）2 <$$>和神经元（<$4）2（<$2）3和（<$7）通过进行灵活的配体对接并与我们自己的NMR数据和其他公开的实验结果（例如，诱变和光亲和标记）;和（c）在对接麻醉剂存在下，平行于（a）重复MD模拟。研究假设：（1）两亲性的麻醉剂与两个相邻的TM 2亚基之间的界面特异性地相互作用，并在疏水带附近的水孔和EC-TM界面的驱动点处相互作用，（2）麻醉剂介导了其中一个驱动点处的疏水性错配，（3）麻醉剂的作用在延长的MD平衡后表现为三级和四级结构水平的整体变化;（4）麻醉剂与EC-TM界面驱动点的相互作用改变了TM 2结构域的RMS波动，而在孔内疏水环带处的相互作用深刻地影响了疏水侧链的重新取向和水通道的闪烁。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The ultimate goal of our study is to understand the molecular mechanisms of general anesthesia. The immediate objective of our computational efforts is to elicit the molecular details of protein motions that are difficult to observe directly with experimental methods but are potentially crucial to the understanding of anesthetic actions on the channel proteins. We will focus on nAChRs that have been suggested to be potential targets for general anesthetics. The specific aims for the study include (a) to perform large-scale MD simulations (10 ns or longer as needed, 3 repeats with different seeds) on open-channel structures of the (¿1)2¿1¿¿ and the neuronal (¿4)2(¿2)3 and (¿7)5 nAChRs in a fully hydrated membrane patch of 3:1:1 POPC:POPA:Cholesterol in the absence of anesthetics (control systems); (b) to identify anesthetic (halothane and isoflurane) interaction sites in the Torpedo (¿1)2¿1¿¿ and the neuronal (¿4)2(¿2)3 and (¿7)5 nAChRs in the open-channel state by performing flexible ligand docking and comparing with our own NMR data and with other published experimental results (e.g., mutagenesis and photoaffinity labeling); and (c) To repeat MD simulations parallel to (a) in the presence of the docked anesthetics. Hypotheses for the study: (1) anesthetics, being amphipathic, interact specifically with the interface between two adjacent TM2 subunits near the hydrophobic girdle in the aqueous pore and at the actuation points at EC-TM interface; (2) anesthetics mediate the hydrophobicity mismatch at one of the actuation points; (3) anesthetic effects are encoded as global changes at tertiary and quaternary structural level after extended MD equilibration; (4) anesthetic interaction with the actuation points at EC-TM interface alters the RMS fluctuation of the TM2 domains, and the interaction at the hydrophobic girdle within the pore profoundly affects the re-orientation of the hydrophobic side chains and the flickering of water passage.