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: 8534429
• 负责人: J. KENT BLASIE
• 金额: $ 29.6万
• 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 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8534429
• 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，我们将研究麻醉剂结合如何干扰机电机制， 偶联麻醉敏感的原型电压门控钠和钾通道。目标1关注 原核Nav-channels NaChBac和NavAb具有优势跨膜结构域。目的2 涉及表达为仅包含其跨膜结构域的eul真核Shaw 2 KV-通道，以及 全长Shaw 2及其与Kv1.2的嵌合体，与Project 2（Covarrubias）合作。同步加速器X射线 &中子干涉技术将应用于单一的，重组磷脂膜 在电化学电池内含有矢量定向的Nav-channel或Kv-channel蛋白，以探测 作为所施加的跨膜电势（电压）的函数的通道的轮廓结构。 X射线干涉测量实验将是“时间分辨的”，每个实验的连续时间范围小于-1毫秒， 提供10-30帧的通道的动力学响应的去极化逐步变化， 电位，从而对生理上的通道的轮廓结构的变化敏感。 通道激活的相关时间尺度与随后的孔开放分开。中子 干涉测量实验将在每个电势处是“稳态的”，或者部分地“时间分辨的”， 每个16 ms的串行时间帧，仅对通道响应的前半部分和后半部分进行采样， 去极化电位的逐步变化。X射线干涉测量实验还将确定 麻醉剂结合位点的定位及其在通道轮廓结构内的占据， 几种典型的挥发性麻醉剂的重卤素原子。结构的空间分辨率 通过定点标记，在X射线中使用重共振原子， 情况下使用氘，中子情况下使用氘，以实现优于± 1 A的位置精度。这些 研究应该为理解麻醉剂结合如何改变 机电耦合的机制，与分子的理论预测比较， 项目4的动力学计算机模拟。