Molecular mechanisms of Tandem Pore potassium channel gating and regulation

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.