Mechanisms of Volatile Anesthetic Modulation of Tandem Pore Potassium Channels

挥发性麻醉剂(VA)可迅速诱导和可逆的无意识状态，这些状态 对于全身麻醉的分娩至关重要，但也会引起全身深度低血压、抑郁 呼吸驱动，以及许多其他增加发病或死亡风险的有害生理干扰 手术期间的死亡率。串联孔(K2P)离子通道是VA响应性钾泄漏通道 是人类基本生理学的基础，也是VA某些有害影响的可疑媒介。 这次K08提案，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 VAS调节K2P通道的分子机制。在Aim1中，我将定义分子 VA与K2P通道结合的决定因素，利用卤醚VAS异氟醚的光亲和类似物和 七氟醚。我将在两个VA敏感的K2P通道(TREK1和TASK1)和 探讨TRAAK K2P通道VA不敏感的分子基础。照片标记结果将是 通过在已鉴定的麻醉剂结合部位残基上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 以光标记和功能研究为指导的动力学模拟将确定预测的额外残基 有助于VA的绑定。在AIM2中，我将确定VA结合改变K2P的机制 构象以实现门控。将利用TREK1的单颗粒冷冻电子显微镜来探索 VA‘s对K2P构象状态的影响，并将首次对K2P-C-进行结构表征。 已知的末端结构域调节VAS和许多其他K2P调节剂的调节效应。通过利用 一种能解决K2P开孔动力学变化的快速混合停流荧光法 通过季铵离子的阻挡，我将考察周围离子组成的组合效应， 脂环境、VAS等TREK1调节剂对TREK1细胞内孔结构的影响，提供 功能语境对我们的结构研究。通过研究K2P相互作用的生物物理基础 通道和VA的，我希望为翻译研究离子通道结构的职业奠定基础， 将其功能和药理作用转化为有意义的临床干预措施，从而提高麻醉护理的安全性。我的 K08研究导师克里娜·尼米根博士是研究离子通道生物物理学和 非常适合在本研究项目的执行过程中提供指导和指导。这个 与麻醉作用机制有关的离子通道药理学和生物物理学的研究 是威尔康奈尔麻醉部历史上感兴趣的焦点，该部门和 体制环境提供了一个出色的背景，使拟议的 学习、提供资源、受保护的研究时间和丰富智力的环境 众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.