Molecular mechanisms of Tandem Pore potassium channel gating and regulation

摘要 本项目的目标是确定控制串联孔活性的分子机制 (K2P)钾通道家族，重点是K2 Ps如何整合各种传入信号， 调节通道功能。K2 P是主要负责产生背景“泄漏”电流的离子通道 设定细胞静息膜电位。K2 P活性的调节直接影响细胞兴奋性， K2 P通道在心脏、神经元、内分泌和血管生物学中发挥重要作用。 K2 Ps的机械敏感性亚家族是该建议的焦点，已被确定为潜在的 药物开发的目标是治疗心律失常，抑郁症和慢性疼痛，虽然努力， 开发靶向K2 Ps的小分子调节剂在很大程度上未能产生高亲和力和亚型 选择剂。同时，已知内源性脂质调节许多K2 P通道，并表现出强的 亚型特异性本提案的总体目标是研究K2 P调节的基础生物学 脂质和膜张力的相关影响，长期目标是利用这些知识来开发 开发针对K2 P通道的改进药理学的框架。为了实现这些目标，我们将 追求多方面的方法，包括cryoEM结构研究，本地质谱，以确定 K2 P/脂质相互作用和K2 P行为的电生理功能研究。我们的第一个目标是检查 正和负变构膜磷脂或游离脂肪酸被感知的机制 通过K2 P通道，重点是K2 P结构中脂质结合位点的分子细节。我们还将 解决变构脂质和膜张力对K2 P门控行为的相互影响的基础。 在aim 2中，我们将探索脂质、机械敏感性和其他K2 P输入信号控制的机制。 在离子传导钾选择性过滤器处的通道输出，限定了通道内的分子连接性。 K2 P通道的结构。综上所述，我们的研究将提供一个详细的结构模型 K2 P门控和调制的研究与这个重要的通道家族的基础生物学广泛相关。

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.