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: 9068185
• 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/9068185
• 关键词: 3-Dimensional; Adverse effects; Anesthetics; Binding; Binding Sites; Cells; Chemosensitization; Chimera organism; Collaborations; Complication; Computer Simulation; Coupling; Cysteine; Deuterium; Drug Design; 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; new therapeutic target; novel therapeutics; 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扰乱机电机制 在麻醉剂敏感的典型电压门控钠和钾通道上偶联。目标1关注的问题 原核细胞的NaV通道NaChBac和Navab具有优势的跨膜区。目标2 涉及真核表达的Shaw2Kv通道只包含其跨膜结构域，以及 全长Shaw 2及其与Kv1.2的嵌合体，与项目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、重组的磷脂膜 在电化学池中包含以矢量为导向的Nav通道或Kv通道蛋白以进行探测 作为施加的跨膜电位(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帧都小于-1ms， 在通道的动力学响应上提供10-30帧，以响应 电位，从而对生理上的通道的轮廓结构的变化敏感 相应的通道激活时间尺度与随后的孔洞打开分开。中子 干涉测量实验在每个电势下都是“稳态”的，或者部分“时间分辨”的。 每个16ms的连续时间帧，仅采样通道响应的前一半和后一半 去极化的电势的逐步变化。X射线干涉测量实验也将确定 麻醉剂结合部位的定位及其在通道轮廓结构中的占有率，利用 几种典型挥发性麻醉药的重卤素原子。结构的空间分辨率 利用x射线中的重共振态原子，通过定点标记将大大加强研究。 在中子情况下，为了达到比1A更好的定位精度，这些 研究应该为理解麻醉剂结合如何改变 机电耦合机理与分子理论预测的比较 项目4的动力学计算机模拟。