X-ray and Neutron Interferometry Studies of Anesthetic Interactions with Voltage

麻醉与电压相互作用的 X 射线和中子干涉研究

• 批准号: 8902162
• 负责人: J. KENT BLASIE
• 金额: $ 29.49万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8902162
• 关键词: 3-Dimensional; Adverse effects; Anesthetics; Binding; Binding Sites; Cells; Chemosensitization; Chimera organism; Collaborations; Complication; Computer Simulation; Coupling; Cysteine; Deuterium; Drug Design; Drug Targeting; Event; General Anesthesia; Goals; Halogens; In Situ; Interferometry; Investigation; Isoflurane; Kinetics; Kv1.2' channel; Label; Length; Membrane; Metals; Molecular; Neutrons; Phospholipids; Photoaffinity Labels; Potassium Channel; Proteins; Resolution; Roentgen Rays; Sampling; Site; Sodium Channel; Structure; Synchrotrons; System; Techniques; Time; Transmembrane Domain; base; design; interest; macromolecule; millisecond; molecular dynamics; mutant; protein structure; reconstitution; research study; response; sensor; sevoflurane; simulation; synchrotron radiation; voltage

In Project 5, we will investigate how anesthetic binding perturbs the mechanism of electromechanical coupling in anesthetic-sensitive, prototypical voltage-gated sodium and potassium channels. Aim 1 concerns the prokaryotic Nav-channels NaChBac and NavAb possessing a dominant transmembrane domain. Aim 2 concerns the eul<aryotic Shaw2 Kv-channel expressed to contain only its transmembrane domain, as well as full-length Shaw 2 and its chimeras with Kv1.2, in collaboration with Project 2 (Covarrubias). Synchrotron xray & neutron interferometry techniques will be applied to single, reconstituted phospholipid membranes containing the vectorially-oriented Nav-channel or Kv-channel protein within an electrochemical cell to probe the profile structure ofthe channel as a function ofthe applied transmembrane electric potential (voltage). The x-ray interferometry experiments will be "time-resolved" with serial time-frames of less than -1ms each, providing 10-30 frames over the channel's kinetic response to the depolarizing step-wise change in the potential, and thereby sensitive to changes in the profile structure ofthe channel on the physiologically relevant time-scales of channel activation separated from subsequent pore opening. The neutron interferometry experiments will be either "steady-state" at each potential, or partially "time-resolved" with serial time-frames of 16ms each, sampling only the first and second halves ofthe channel's response to the depolarizing step-wise change in the potential. The x-ray interferometry experiments will also determine the localization of anesthetic binding sites and their occupancy within the channel's profile structure, utilizng the heavy halogen atoms of several prototypical volatile anesthetics. The spatial resolution ofthe structural studies will be substantially enhanced through site-directed labeling, with heavy resonant atoms in the x-ray case and deuterium in the neutron case, to achieve a the positional accuracy of better than 1 A. These studies should provide an experimental structural basis for understanding how anesthetic binding alters the mechanism of electromechanical coupling, for comparison with the theoretical predictions from the molecular dynamics computer simulations of Project 4.

在项目5中，我们将研究麻醉剂的结合如何消除机电机制 在麻醉敏感的，典型的电压门控钠和钾通道中耦合。目标1关注 原核Nav-nacnels Nachbac和Navab具有主要的跨膜结构域。目标2 关注Eul <aryotic shaw2 kV通道，该通道仅包含其跨膜域，以及 全长肖2及其与KV1.2的嵌合体与项目2（Covarrubias）合作。 Synchrotron XRARE 和中子干涉测量技术将应用于单一重构的磷脂膜 在电化学细胞中包含以载体为导向的NAV通道或KV通道蛋白 通道的轮廓结构是应用的跨膜电势（电压）的函数。 X射线干涉测量实验将“时间分辨”，串行时间框架每个小于-1ms， 在通道对去极化的逐步变化的动力学响应上提供10-30帧 潜力，从而敏感到生理上通道的轮廓结构的变化 通道激活的相关时间尺度与随后的孔口开口分开。中子 干涉测量实验要么在每个电势上是“稳态”，要么部分地“时间分辨”与 每个16ms的串行时间框架，仅对频道对频道响应的第一和第二半采样 去极化的电势变化。 X射线干涉法实验还将确定 麻醉结合位点的定位及其在通道的轮廓结构中的占用率 几种原型挥发性麻醉药的重卤素原子。结构的空间分辨率 研究将通过定向标记实质上增强，并在X射线中具有沉重的谐振原子 在中子情况下，案例和氘，以达到比1 A的位置准确性更好。 研究应提供实验结构基础，以了解麻醉结合如何改变 机电耦合的机理，以与分子的理论预测进行比较 项目4的动态计算机模拟。