PROJECT SUMMARY: Significance: To date, no targeted therapies exist that prevent or improve the outcomes of patients with Acute Respiratory Distress Syndrome (ARDS). Despite an increasing awareness of hyperoxia (HO)-induced lung injury and low tidal volume mechanical ventilation (MVL) strategies, mortality rates have plateaued at 35-45% causing 75,000 deaths/year, and suggesting that we may have maximized the therapeutic potential of these standard interventions. Additionally, progress in this field is hindered by the technical difficulties in experimentally replicating the inflammatory environment at the alveolar-capillary interface with current ARDS models. Therefore, the identification of molecular targets, and the development of new therapeutic strategies and improved ARDS models represents a high priority topic with substantial impact in Pulmonary and Critical Care Medicine. In search for new therapeutic approaches, the Schwingshackl laboratory discovered epithelial TREK-1 K+ channels as key regulators of alveolar inflammation using simple, one-hit HO- and Influenza-A virus (IAV)- induced lung injury models. However, the importance and protective potential of TREK-1 K+ channels in clinically more relevant triple hit (IAV+HO+MVL) ARDS models remains unknown. To bridge these knowledge gaps and advance the field in a new direction, based on strong and exciting new preliminary data the authors now hypothesize that (i) ARDS decreases epithelial and endothelial TREK-1 levels, which causes cell membrane depolarization and subsequently promotes Ca2+-dependent alveolar inflammation, and (ii) this injurious cascade can be counteracted by activation of the subset of residual TREK-1 channels. Using IAV as the most common ARDS trigger worldwide (besides SARS-CoV-2), and clinically-relevant HO and MVL strategies, Aim 1 will investigate whether IAV, alone and combined with HO/MVL, decreases epithelial and endothelial TREK-1 levels, and thus accelerates further lung injury. Aim 2 will determine whether activation of the subset of residual TREK-1 channels can protect against IAV-induced ARDS. Aim 3 will establish the molecular signaling mechanisms and protein-protein interaction networks underlying TREK-1-mediated protection, by using high-throughput electrophysiological approaches and precision medicine Functional Enrichment Analysis tools, to ultimately facilitate rational drug design. For these studies, the authors will utilize novel and complementary pharmacological and genetic loss- and gain-of-function approaches, including newly- developed TREK-1 activating compounds (ML335, ML6733, BL1249), targeted cell type-specific TREK-1 overexpressing and TREK-1 KO mouse models, primary human alveolar epithelial/endothelial cells and lung tissue, and two next-generation experimental ARDS models (Bioreactor; modified microfluidic SynVivo platform). Potential for high Impact: (i) Establish aberrant TREK-1 signaling as an unrecognized pathway in ARDS; (ii) hig...