Mechanisms of Volatile Anesthetic Modulation of Tandem Pore Potassium Channels

挥发性麻醉剂（VA）可产生快速诱导和可逆的无意识状态， 对于分娩至关重要的全身麻醉，也会引起全身性深度低血压、抑郁 呼吸驱动，以及许多其他有害的生理扰动，增加发病率的风险，或 手术期间死亡。串联孔（K2 P）离子通道是VA响应性钾渗漏通道， 是基本人体生理学的基础，并且是VA的某些有害作用的疑似介质。在 这个K 08的建议，我将利用我在离子通道生理学和生物物理学领域的背景来探索 VA调节K2 P通道的分子机制。在目标1中，我将定义分子 VA与K2 P通道结合的决定因素，利用卤醚VA的光亲和类似物异氟烷和 七氟烷我将在两个VA敏感性K2 P通道（TREK 1和TASK 1）中鉴定VA结合位点， 探索TRAAK K2 P通道VA不敏感性的分子基础。光标记结果将是 通过在确定的麻醉剂结合位点残基处引入突变来验证功能相关性， 进行功能研究，以评估门控行为或麻醉敏感性的改变。分子 由光标记和功能研究指导的动力学模拟将确定预测 有助于VA结合。在Aim 2中，我将确定VA结合改变K2 P的机制。 以实现门控。将利用TREK 1的单颗粒冷冻电子显微镜来探索 VA对K2 P构象状态的影响，并将产生K2 P C-的第一个结构表征 K2 P是已知调节VA和许多其他K2 P调节剂的调节作用的末端结构域。通过利用 一种能够解析K2 P开孔动力学变化的快速混合停流荧光测定法 通过季铵离子的阻断，我将研究周围离子组成的组合效应， 脂质环境、VA和其他TREK 1调节剂对TREK 1的细胞内孔结构的影响， 我们的结构研究的功能背景。通过研究K2 P和K2 P之间相互作用的生物物理基础， 通道和VA的，我希望奠定基础的职业翻译研究的离子通道结构， 功能和药理学转化为有意义的临床干预措施，提高麻醉护理的安全性。我 K 08研究导师Crina Nimigean博士是离子通道生物物理学研究的国际专家， 非常适合在执行本研究项目期间提供指导和指导。的 离子通道药理学和生物物理学与麻醉作用机制有关研究， 一直是威尔康奈尔大学麻醉系的历史焦点， 体制环境为成功完成拟议的 研究，提供资源，受保护的研究时间，以及丰富的智力环境， 许多可用的顾问和潜在的合作者。

Volatile anesthetic (VA) agents produce rapidly inducible and reversible states of unconsciousness that are vital for the delivery of general anesthesia, but also cause profound systemic hypotension, depressed respiratory drive, and numerous other deleterious physiological perturbations that increase risks of morbidity or mortality during surgery. Tandem pore (K2P) ion channels are VA responsive potassium leak channels that are fundamental to basic human physiology and suspected mediators of some deleterious effects of VA’s. In this K08 proposal, I will utilize my background in the field of ion channel physiology and biophysics to explore the molecular mechanism by which VAs modulate K2P channels. In Aim1, I will define the molecular determinants of VA binding to K2P channels, utilizing photoaffinity analogs of the haloether VAs isoflurane and sevoflurane. I will identify VA binding sites in two VA sensitive K2P channels (TREK1 and TASK1) and explore the molecular basis for the VA insensitivity of TRAAK K2P channels. Photolabeling results will be verified for functional relevance by introducing mutations at identified anesthetic binding site residues and performing functional studies to assess for altered gating behavior or anesthetic sensitivity. Molecular dynamics simulation guided by photolabeling and functional studies will identify additional residues predicted to contribute to VA binding. In Aim2, I will determine the mechanism by which VA binding alters K2P conformation to effect gating. Single particle cryo-electron microscopy of TREK1 will be utilized to explore the effects of VA’s on K2P conformational state and will produce the first structural characterization of the K2P C- terminal domain known to regulate the modulatory effects of VAs and many other K2P modulators. By utilizing a rapid mixing stopped flow fluorometric assay capable to resolving changes in the kinetics of K2P open pore block by quaternary ammonium ions, I will examine the combinatorial effects of surrounding ionic composition, lipid environment, VAs and other TREK1 modulators on the intracellular pore structure of TREK1, providing functional context to our structural studies. By studying the biophysical basis for the interaction between K2P channels and VA’s, I hope to lay the groundwork for a career translating studies of ion channel structure, function and pharmacology into meaningful clinical interventions that improve the safety of anesthetic care. My K08 research mentor, Dr. Crina Nimigean, is an international expert in the study of ion channel biophysics and is extremely well suited to provide mentorship and guidance during the execution of this research project. The study of ion channel pharmacology and biophysics as they pertain to mechanisms of anesthetic action has been a historical focus of interest in the Weill Cornell Department of Anesthesia and both the departmental and institutional environments provide an outstanding backdrop to enable successful completion of the proposed studies, providing resources, protected research time, and an intellectually enriching environment with numerous available advisors and potential collaborators.