In the brain, KCNQ2 ? KCNQ5 co-assemble to form the M-channel, which regulates neuronal excitability and is a high-impact therapeutic target in several mental disorders, including Major Depressive Disorder, Schizophrenia, Bipolar Disorder and Attention-Deficit Hyperactivity Disorder. Physiologically, the M-channel is responsible for dynamic control of the resting membrane potential. Cholinergic signaling through muscarinic receptors results in depletion of plasma membrane PIP2, which in turn results in closure of the M-channel. A direct PIP2 ? channel interaction is required to open the pore domain of KCNQ channels, however the structural basis of this interaction is unknown. This work will determine the activated-state structure of human KCNQ2 bound to calmodulin (CaM) and PIP2 in a lipid environment, using cryo electron microscopy. In a general sense, this structure will expand the understanding of ion channel activation mechanism. In a specific sense, the structure of activated KCNQ2-CaM will be a valuable asset to structure-guided development of KCNQ2 activators, targeting the above-mentioned mental disorders. In addition to structural studies, this proposal seeks to characterize the pharmacology of KCNQ2, elucidating the state-dependent thermodynamics and cooperativity of known KCNQ2 small molecule activators using isothermal titration calorimetry. The interaction enthalpies and entropies measured will report on how these small molecule activators engage their target, KCNQ2, and can inform future drug development efforts to improve the potency of these compounds. Finally, this project will develop and validate direct, in vitro, high-throughput assays for KCNQ2 activators and inhibitors. The assays will be based on a standard liposome flux fluorescence assay (LFFA) that be adapted to study KCNQ2 without and with PIP2 in order to test activators and inhibitors, respectively. These assays will be validated with known KCNQ2 activators and inhibitors, and will significantly expand the current arsenal of KCNQ2 drug screening techniques. In support of these proposed studies, robust biochemical preparations of KCNQ2-CaM and KCNQ1-CaM have been developed, and preliminary cryoEM data on KCNQ1-CaM have been collected. Additionally, the applicant has developed technical expertise in cryoEM while engaged in studies of an archaeal potassium channel, and has conducted LFFAs for a human proton channel. The structural, functional and pharmacological studies proposed here will yield insights into the molecular mechanisms of neuronal function, as well as directly inform future drug development efforts.