PROJECT SUMMARY This project aims to understand the function and regulation of three ion channels that conduct K+ across cell membranes and belong to the two-pore domain K+ channel family. We will apply cryo-electron microscopy to determine structures of the channels in different functional states within lipid environments that mimic the cellular membrane and electrophysiological recordings to characterize their activities in order to derive physical models for channel gating and modulation. We aim to capture structural snapshots of the different open and closed conformations for each channel by varying conditions that alter channel function including solution composition, lipid composition, and presence of small molecules or interacting proteins. The three channels are members of different branches of the two-pore domain K+ channel family. While they share a common structural architecture, each channel is regulated by pH in a different way; one is inhibited by protons on both sides of the membrane, the second is inhibited only by extracellular protons, and the third is both inhibited and altered in its ionic selectivity by extracellular protons. The underlying molecular mechanisms by which pH is sensed and converted into a change in channel activity are correspondingly different between the channels. Each channel is further regulated by a distinct set of factors including signaling lipids, interacting proteins, solution ion composition, and small molecule drugs. Comparative analyses of the three structurally and evolutionarily related K+ channels will therefore provide additional insight into their functional properties and biological roles. Two-pore domain K+ channels mediate cellular electrical signaling by establishing and maintaining the resting membrane potential and opposing excitability. The channels under study here are involved in respiratory regulation, cardiac rhythm generation, blood pressure control, central chemoreception, and systemic pH homeostasis among other processes. Their dysregulation is implicated in cardiac arrythmia, kidney disease, and hypertension in humans and they are targets of anesthetics, antiarrhythmics, and drugs under investigation for obstructive sleep apnea. Therefore, in addition to providing fundamental mechanistic insight into the physical and chemical basis for channel function, this work will serve as a basis for the development of more potent and specific pharmacological agents targeting ion channels to promote health and treat disease. Importantly, the technical and methodological advances developed here for structural characterization of small membrane proteins in lipid environments are expected to be widely applicable and will facilitate insights across the breadth of biology in which membrane proteins play important roles.