PROJECT SUMMARY We have previously identified distinct functional outcomes in lung alveolar septal endothelium on activation of TRPV4, a Ca channel in the vanilloid transient receptor potential family, or the 1G T-type voltage-gated Ca 2+ 2+ channel. Despite equivalent whole-cell Ca2+ transients in the extremely thin septal endothelium, TRPV4 only increases endothelial permeability while the T-channel only increases endothelial surface expression of P- selectin. As a result, we propose a critical paradigm shift, from a global perspective of Ca2+-dependent signaling to one where Ca2+ microdomains, orchestrated by mitochondrial-dependent Ca2+ buffering, are organized to yield discrete functional outcomes within lung microvascular endothelial cells. Our preliminary data suggest discrete localization of these Ca2+channels and distribution of mitochondria even into the attenuated cell periphery in lung microvascular endothelium in situ. Further, we have documented that mitochondrial bioenergetic dysfunction leads to loss of domain constraints with greater spread and duration of TRPV4-mediated Ca2+ transients and increased endothelial permeability in lung microvascular endothelium. Collectively, these observations led us to the HYPOTHESIS that in lung microvascular endothelium, mitochondrial Ca2+ buffering constrains Ca2+ influx via TRPV4 or the T-type channel to spatially delimited cytosolic microdomains yielding specificity of functional outcomes, constraints lost with mitochondrial dysfunction. Our SPECIFIC AIMS are to: 1) determine the contribution of mitochondria to buffering of the spatial spread, dynamics and functional specificity of Ca2+ signals on activation of TRPV4 or T-type Ca2+ channels, and 2) determine the extent to which mitochondrial bioenergetic dysfunction decreases the threshold for and specificity of functional outcomes on activation of TRPV4 or T-type Ca2+ channels. We will utilize innovative high-speed hyperspectral excitation scanning imaging and novel analytical tools to detect and interpret signal dynamics with high spatial and temporal resolution. These data will be interpreted in context of localized functional outcomes, in naïve endothelium, in endothelium after disruption of mitochondrial- dependent buffering and after initiation of mitochondrial bioenergetic dysfunction in the intact lung with hyperoxia and Pseudomonas aeruginosa-induced sepsis. We predict that such dysfunction will lead to blurring of specificity for Ca2+ signaling, altering the set point from which lung endothelium interprets Ca2+ signaling with mechanical stress. This work will provide the first insight into mechanisms underlying Ca2+ microdomains in lung microvascular endothelium. To accomplish this, we have assembled an outstanding team with expertise spanning from structural and functional determinants of endothelial permeability, development and use of novel tools, and modeling of signaling domains/networks to bioenergetics and sepsis.