Role of calcineurin in store-operated calcium entry of pulmonary endothelium

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DESCRIPTION (provided by applicant): Endothelial cells line blood vessels and allow the selective transport of molecules. Disruption of this barrier leads to increased permeability, which can contribute to certain pathologies, including inflammation, acute respiratory syndrome, and athlerosclerosis. Cytosolic calcium entry through store-operated calcium channels (SOC) contributes significantly to barrier disruption. Activation of Isoc, a calcium-selective SOC curren, leads to endothelial cell gap formation. The transient receptor canonical (TRPC) proteins TRPC1 and TRPC4 are part of Isoc channel structure, and TRPC4 is necessary for proper Isoc channel function. When Isoc is activated, the channel opens resulting in calcium influx across the cell membrane. Conversely, channel closure restricts calcium entry. Recently, it was shown that phosphorylation of serine 712 of a TRPC3 channel was responsible for negative regulation of the channel. Unpublished results from our laboratory suggest that inactivation of the Isoc channel is dependent on phosphorylation of TRPC4. It appears that TRPC4 is dephosphorylated when internal calcium stores are released allowing channel activation and is phosphorylated following Isoc channel opening. Understanding the phosphatase involved in readying the channel for activation is crucial to the larger picture of Isoc channel regulation. Th identity and contribution of the phosphatase regulating phosphorylation status of TRPC4 are unknown. Calcineurin (CN) is a calcium/calmodulin activated serine-threonine phosphatase. CN is inhibited by the immunophilin FKBP51-FK506 immunosuppressant drug complex. However, there have been reports of the ability of FKB51 and other FKBP immunophilins to bind to and inhibit CN independently of FK506. FKBP51 is expressed in capillary endothelial cells (PMVECs), but levels of the protein are nearly absent in pulmonary artery endothelial cells (PAECs). PAECs exhibit a higher SOC entry compared with PMVECs. The differential regulation of Isoc channel in PAECs and PMVECs is incompletely understood. FKBP51 may play a role in this differential regulation between the two endothelial cell types. This project tets the hypothesis that calcineurin regulates the dephosphorylation status of TRPC4 on the endothelial Isoc channel and FKBP51 inhibits the ability of calcineurin to dephosphorylate TRPC4, rendering the channel less active.