PROJECT SUMMARY: Significance: Oxygen supplementation (hyperoxia; HO) is the most frequently applied therapy for hospitalized patients and the cornerstone of treatment for acute hypoxic respiratory failure (ARF). It is well known, however, that HO exposure can not only promote existing lung injury but also initiate inflammation and barrier dysfunction in otherwise healthy lungs. The inflammatory response evoked by HO is particularly damaging to alveolar epithelial and endothelial cells causing cellular apoptosis and alveolar barrier disruption. Clinically, the recognition of HO-induced acute lung injury (HALI) led to an increased awareness of oxygen toxicity and efforts to minimize oxygen exposure for ARF patients. Although clinical and experimental studies have identified several potential mechanisms underlying HALI, currently no therapies exist to prevent or counteract HALI, and the length of hospitalization of ARF patients has remained unchanged for two decades. These findings underscore the urgent need for identifying molecular targets to facilitate rational drug design against HALI. In the search for such new targets, we discovered TREK-1 potassium channels as potential new key regulators of HALI. Our preliminary data support the novel hypothesis that HO downregulates epithelial and endothelial TREK-1 channels, which results in cell membrane depolarization, subsequent opening of voltage- gated Ca2+ channels, and as a consequence increased inflammatory mediator secretion, cell apoptosis and alveolar barrier dysfunction. Furthermore, we propose that enhancement of TREK-1 activity can counteract this injurious cascade. We will test this hypothesis in three Specific Aims: In Aim1 we will identify the cell type(-s) predominantly affected by HO-induced TREK-1 downregulation, using epithelial and endothelial cell-specific TREK-1 KO mouse models and primary cells isolated from these mice. In Aim 2 we will determine the protective effects of TREK-1 enhancement against HALI using novel TREK-1 activating compounds, new cell type-specific TREK-1 overexpressing mouse models, and primary epithelial and endothelial cells isolated from these mice. In Aim 3 we will dissect the structural composition and biophysical properties of epithelial and endothelial TREK-1 channels at baseline and under HO conditions, and propose a novel signaling mechanism by which TREK-1 channels could regulate inflammation and barrier dysfunction during HALI. This study will impact the field of acute lung injury by establishing aberrant epithelial and endothelial TREK- 1 signaling in the lung as a previously unrecognized pathway in HALI, and TREK-1 activation as the first targeted therapeutic approach against HALI.