Ether-a-go-go (EAG) potassium selective channels are important regulators of neuronal excitability and cancer progression. Defects in EAG channel function are associated with neurological disorders and cancer. Despite the physiological importance of EAG channels, molecular mechanisms of EAG channel regulation by intracellular ligands and clinically relevant EAG channel regulators are not known. The goal of this proposal is to uncover molecular mechanisms of EAG channel regulation by intracellular ligands recently discovered by our laboratory and to determine a therapeutic potential of these ligands for treatment of diseases linked to EAG channels. In Specific Aim 1 we plan to solve X-ray structures of the intracellular Per-Arnt-Sim (PAS) and cyclic nucleotide- binding homology (CNBH) domains of EAG channels bound to the recently identified ligands and conduct computational simulations of the ligand binding to the PAS and CNBH domains to uncover the structural basis of EAG channel regulation by the intracellular ligands. The structural findings will be then used as a road map to guide functional experiments on the molecular mechanisms of EAG channel regulation by the ligands. In Specific Aim 2 we plan to use surface plasmon resonance method to identify novel EAG channel ligands that affect channel function through PAS and CNBH domain interface. We will then use electrophysiology to determine functional implications of strengthening or weakening of the PAS/CNBH domain interface by the identified regulators on the function of EAG channels. In Specific Aim 3 we plan to use tissue culture and zebrafish xenograft models to test therapeutic potential of the identified regulators for treatment of cancer. The results of these studies will be crucial for understanding fundamental regulatory mechanisms of EAG and related ERG and ELK channels, and for attaining therapeutic potential of EAG channel regulators.