Lung endothelium forms a semi-permeable barrier that restricts water, solute and macromolecular access to the interstitium, which is important to optimize gas exchange. Inflammation disrupts this barrier function, causing accumulation of a protein-rich fluid in interstitial and alveolar compartments that contributes to the genesis of acute respiratory distress syndrome. Inflammatory mediators disrupt the endothelial barrier by promoting calcium entry across the cell membrane, through store operated calcium (SOC) entry channels. This calcium transition triggers cytoskeletal reorganization that initiates inter-cellular gap formation, and increases permeability. SOC entry occurs through both calcium selective (IS0C) and non-selective channels, although IS0C activation is sufficient to increase lung endothelial cell permeability. Canonical transient receptor proteins 1 (TRPC1) and 4 (TRPC4) contribute subunits to the channel that underlies the IS0C, although the precise TRPC1/4 subunit composition remains undetermined. IS0C activation requires a highly specific interaction between TRPC4 and the membrane skeleton, suggesting channel activation is TRPC4-dependent and not TRPC1-dependent. Indeed, TRPC4 interacts with protein 4.1, and protein 4.1 tethers the channel to the spectrin membrane skeleton. Interestingly, protein 4.1 binds to spectrin just 21 residues downstream from its filamentous (f)-actin-binding domain. Calcium that permeates the IS0C may disrupt the spectrin-f-actin interaction, initiating the cytoskeletal reorganization necessary for endothelial cell gap formation. Thus, this proposal tests the overall HYPOTHESIS that TRPC1 and TRPC4 contribute subunits that form the molecular basis of the IS0C, which provides a calcium source that disrupts spectrin-f-actin binding necessary to increase endothelial permeability.
Specific aims test the related hypotheses that: [1] TRPC1 and TRPC4 each contribute subunits needed to generate the IS0C; [2] protein 4.1 interacts directly with its binding domain on TRPC4, necessary to activate the IS0C; and, [3] calcium transitions through the /S0C are sufficient to disrupt the spectrin-f-actin association, and increase endothelial cell permeability. Studies proposed in this application are significant because they may reveal the molecular composition and regulation of an ion channel that specifically controls endothelial cell barrier function, providing a potentially novel pharmacological target for therapy in inflammatory lung disease. ? ? ?
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