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: 8740499
• 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
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8740499
• 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 麻醉敏感的原型电压门控钠和钾通道中的耦合。目标 1 关注点 原核生物 Nav 通道 NaChBac 和 NavAb 拥有显性跨膜结构域。目标2 涉及表达为仅包含其跨膜结构域的 eul<aryotic Shaw2 Kv 通道，以及 与 Project 2 (Covarrubias) 合作，制作全长 Shaw 2 及其与 Kv1.2 的嵌合体。同步加速器X射线 & 中子干涉测量技术将应用于单一的、重构的磷脂膜 电化学电池内含有矢量导向的 Nav 通道或 Kv 通道蛋白以进行探测 通道的轮廓结构作为所施加的跨膜电势（电压）的函数。 X射线干涉测量实验将是“时间分辨”的，每个串行时间帧小于-1毫秒， 提供 10-30 帧的通道动态响应，以消除偏振的逐步变化 电位，从而对生理上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 干涉测量实验要么在每个电位下都是“稳态”，要么是部分“时间分辨”的 每个 16ms 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中的占据，利用 几种典型的挥发性麻醉剂的重卤素原子。结构空间分辨率 通过 X 射线中的重共振原子进行定点标记，研究将得到实质性加强 在中子情况下和氘情况下，实现优于1 A的定位精度。这些 研究应该为理解麻醉剂结合如何改变 机电耦合机制，与分子理论预测进行比较 项目 4 的动力学计算机模拟。