Molecular mechanisms of Tandem Pore potassium channel gating and regulation

摘要 这个项目的目标是确定控制串联孔活动的分子机制。 (K2P)钾通道家族，重点介绍K2P如何整合一组不同的传入信号来 调节经络功能。K2P是主要负责产生背景“泄漏”电流的离子通道 这设置了细胞静息膜的电位。K2P活性的调节直接影响细胞的兴奋性和 K2P通道在心脏、神经、内分泌和血管生物学中发挥重要作用。 作为这一提议的焦点的K2P的机械敏感亚家族已经被确定为潜在的 治疗心律失常、抑郁和慢性疼痛的药物开发目标，尽管努力 开发针对K2P的小分子调节剂在很大程度上未能产生高亲和力和亚型 有选择性的药物。同时，已知内源性脂质调节许多K2P通道，并表现出很强的 亚型特异性。这项建议的总体目标是研究K2P调节的基本生物学 通过脂质和膜张力的相关影响，长期目标是利用这一知识来开发 开发针对K2P通道的改进药理学的框架。为了实现这些目标，我们将 采用多方面的方法，包括低温EM结构研究、天然质谱学来定义 K2P/脂质相互作用，以及K2P行为的电生理功能研究。我们的首要目标是研究 正负变构膜磷脂或游离脂肪酸的感知机制 通过K2P通道，重点研究K2P结构中脂质结合部位的分子细节。我们还将 阐述变构脂质和膜张力对K2P门控行为相互影响的基础。 在AIM2中，我们将探索脂类、机械敏感性和其他K2P输入信号控制的机制 离子导电钾选择性过滤器的通道输出，定义了 K2P航道的结构架构。总而言之，我们的研究将提供一个详细的结构模型 这与这一重要的通道家族的基本生物学广泛相关。

Abstract The goals of this project are to determine the molecular mechanisms that control activity of the tandem pore (K2P) family of potassium channels, with a focus on how K2Ps integrate a diverse set of incoming signals to regulate channel function. K2Ps are ion channels primarily responsible for producing background “leak” currents that set cellular resting membrane potential. Modulation of K2P activity directly affects cellular excitability and K2P channels have been implicated to play important roles in cardiac, neuronal, endocrine, and vascular biology. The mechanosensitive subfamily of K2Ps that are the focus of this proposal have been identified as potential drug development targets for treatment of cardiac arrhythmia, depression, and chronic pain, though efforts to develop small molecule modulators that target K2Ps have largely failed to produce high affinity and subtype selective agents. Meanwhile, endogenous lipids are known to modulate many K2P channels and show strong subtype specificity. The overall aim of this proposal is to examine the basic biology underlying K2P modulation by lipids and the related effects of membrane tension, with the long-term goal of using this knowledge to develop a framework for development of improved pharmacology against K2P channels. To achieve these goals, we will pursue a multifaceted approach that includes cryoEM structural studies, native mass spectrometry to define K2P/lipid interactions, and electrophysiological functional studies of K2P behavior. Our first aim will examine the mechanisms by which positive and negative allosteric membrane phosopholipids or free fatty acids are sensed by K2P channels, with a focus on the molecular details of lipid binding sites in the K2P structure. We will also address the basis for the interrelated impacts of allosteric lipids and membrane tension on K2P gating behavior. In aim2, we will explore the mechanism by which lipids, mechanosensitivity, and other K2P input signals control channel output at the ion conducting potassium selectivity filter, defining the molecular connectivity within the structural architecture of the K2P channel. Taken together, our studies will provide a detailed structural model of K2P gating and modulation that is broadly relevant to the basic biology of this important family of channels.