Mechanisms Underlying Small Molecule Regulation of K2P Channel Activity for the Treatment of Pain

小分子选择性上调K2P通道活性是治疗疼痛的一种有吸引力的策略。这名学生将参与一项关于小分子和其他生理上相关的配体如何调节K2P通道活动的主要研究。我们最近确定了两个典型的人类K2P通道(TREK1和TREK2)在不同构象和与状态依赖的抑制剂的复合体中的晶体结构。这对K2P通道如何工作提供了重要的见解，现在提出了一个令人兴奋的实验框架来解决关于K2P通道门控和调节的分子机制的更复杂的问题。我们目前正在结合膜蛋白结构生物学的一些最新进展(LCP和X射线自由电子激光)来鉴定TREK1和TREK2通道的新构象，以及它们与一些(溴化)小分子结合剂(包括抑制剂和激活剂)的络合物的结构。基于这些信息，学生将学习以下多学科方法：1)通过定点突变和功能分析验证这些配体结合位点。2)剖析配体作用的结构和生物物理机制。3)通过合理设计/合成新型配体来提高/调节配体的效力。4)研究新型配体在不同K2P通道中的选择性，并在模型细胞系统(如培养的大鼠背根神经节和/或人类干细胞衍生的感觉神经元)中测试它们的效力。这些目标将涉及对异源表达的WT和突变通道的电生理分析。这将使我们能够探索配体结合如何影响通道门控和调节，以及不同的激活剂如何影响状态依赖阻滞剂的抑制。目前的结果已经确定了结构上不同的开放状态，拟议的研究将使我们能够将这些不同的状态与药物作用机制联系起来。我们还将利用我们与业界的合作，通过合理设计和合成新的配体和高通量筛选配体文库来提高配体的效率。配基的有效性和选择性也将通过研究它们对不同克隆的K2P通道(例如Trek/TRAAK/Tresk)以及对模型细胞类型(例如DRG和HES神经元)中天然K2P通道电流的影响来检验，在这些模型细胞中，主要的K2P通道被认为是TREK2和Tresk。至于所涉及的潜在风险，我们认为，由于这一项目是一个更大的既定和成功的研究方案的一部分，而且我们有大量已发表的初步证据表明拟议方法的可行性，这些风险已经得到缓解。

The selective up-regulation of K2P channel activity by small molecules represents an attractive strategy for the treatment of pain. The student will be part of a major study of how small molecules and other physiologically relevant ligands regulate K2P channel activity. We have recently determined the crystal structures of two prototypical human K2P channels (TREK1 and TREK2) in different conformations and in complex with a state-dependent inhibitor. This has provided important insights into how K2P channels work, and now presents an exciting experimental framework to address more complex questions about the molecular mechanisms of K2P channel gating and regulation. We are currently combining some of the most recent advances in membrane protein structural biology (LCP and X-Ray Free Electron Lasers) to identify novel conformations of TREK1 and TREK2 channels and also their structures in complex with a number of (brominated) small molecule binders (both inhibitors and activators). Based upon this information, the student will be involved in a multidisciplinary approach to: 1) Validate these ligand binding sites via site-directed mutagenesis and functional analysis.2) Dissect the structural and biophysical mechanisms underlying ligand action.3) Improve/modulate ligand efficacy through the rational design/synthesis of novel ligands.4) Examine the selectivity of novel ligands amongst different K2P channels, and test their efficacy in model cellular systems (e.g. cultured rat DRG and/or human stem cell-derived sensory neurons). These aims will involve electrophysiological analysis of heterologously expressed WT and mutant channels. This will allow us to probe how ligand binding affects channel gating and regulation, and how different activators influence inhibition by state-dependent blockers. Current results have identified structurally distinct open states and the proposed studies will allow us to correlate these different states with mechanisms of drug action. We will also exploit our collaborations with industry to improve ligand efficacy through the rational design and synthesis of novel ligands and high throughput-screening of ligand libraries. Ligand efficacy and selectivity will also be examined by studying their effects on different cloned K2P channels (e.g. TREK/TRAAK/TRESK), as well as on native K2P channel currents in model cell types e.g. DRG and hES neurons where the principal K2P channels are thought to be TREK2 and TRESK. With regards to potential risks involved, we believe they are mitigated by the fact this project forms part of a larger established and successful research programme, and because we have extensive published and preliminary evidence demonstrating the feasibility of the proposed methods.