PROJECT SUMMARY ATP-sensitive potassium (KATP) channels, gated by intracellular nucleotides ATP and ADP, couple cell energetics with membrane excitability to govern a wide range of physiological processes vital to energy homeostasis. KATP channels are unique hetero-octameric membrane protein complexes of four inward rectifier K+ channel (Kir6.1 or Kir6.2) subunits and four sulfonylurea receptor (SUR1, SUR2A, or SUR2B) subunits. Various Kir6.x/SURx combinations generate KATP channel isoforms with distinct tissue distribution, nucleotide sensitivity, and pharmacology. The most prominent KATP channels are those of Kir6.2/SUR1, Kir6.2/SUR2A, and Kir6.1/SUR2B combinations, representing the major pancreatic, cardiac, and vascular smooth muscle isoforms, respectively. Genetic mutations in the various KATP channels underlie a number of endocrine, cardiovascular, and neuronal, and muscular diseases, as exemplified by Cantú syndrome, a severe pleiotropic systemic hypotension disorder caused by gain of function mutations in the vascular Kir6.1/SUR2B KATP channel. A central goal in the KATP channel field is to understand the structure-function relationship of KATP channel isoforms in order to develop mechanism-based, isoform-specific therapies for disease caused by KATP channel dysfunction. A key barrier to progress has been a lack of high resolution channel structures. Recently, we have broken this barrier by resolving 3D structures of the pancreatic Kir6.2/SUR1 channel to near atomic resolutions using cryoEM. In this new application, we seek to carry this momentum and determine structures of the other KATP channel isoforms. We hypothesize that comparing and contrasting related KATP channel complexes will reveal the general design principles that allow KATP channels to operate as ATP/ADP sensors and the specific mechanisms that underlie the unique gating properties and pharmacology of different KATP channel isoforms. We will test the hypothesis using a multipronged approach that combines single-particle cryoEM, molecular dynamics simulations, in silico compound screening, and functional assays in three independent but integrated Specific Aims. (1) Determine cryoEM structures and conformational dynamics of KATP channel isoforms in apo, inhibitors-bound and activators- bound states. (2) Test mechanistic hypotheses on KATP channel isoform-specific biophysical properties, nucleotides sensitivities, and pharmacology. (3) Conduct proof-of-concept structure-based drug discovery studies towards targeted pharmacology for Cantú mutations in vascular KATP channels. The scientific premise of the proposal is built on a wealth of KATP channel literature and rigorous preliminary and published studies from the applicant and her co-investigator and collaborators. The proposal is innovative as it aims to generate new structures, establish new concepts in KATP gating regulation mechanisms, and discover new vascular KATP channel inhibitors for Cantú patients. Successful outcom...