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

描述（由申请人提供）：内皮细胞排列在血管中，允许分子的选择性转运。这一屏障的破坏导致渗透性增加， 可导致某些病理，包括炎症、急性呼吸道综合征和运动失调。胞浆钙通过钙库操纵的钙通道（SOC）进入，对屏障破坏有重要作用。Isoc（一种钙选择性SOC电流）的激活导致内皮细胞间隙的形成。瞬时受体典型（TRPC）蛋白TRPC 1和TRPC 4是Isoc通道结构的一部分，TRPC 4是正确的Isoc通道功能所必需的。当Isoc被激活时，通道打开，导致钙穿过细胞膜流入。相反，通道关闭限制钙进入。最近，它表明，磷酸化的丝氨酸712的TRPC 3通道是负责负调控的通道。我们实验室未发表的结果表明，Isoc通道的失活依赖于TRPC 4的磷酸化。似乎TRPC 4在释放内部钙储存允许通道活化时被去磷酸化，并且在Isoc通道开放后被磷酸化。了解参与准备激活通道的磷酸酶对Isoc通道调控的大局至关重要。调节TRPC 4磷酸化状态的磷酸酶的身份和贡献是未知的。钙调神经磷酸酶（CN）是一种钙/钙调蛋白激活的丝氨酸-苏氨酸磷酸酶。CN被亲免素FKBP 51-FK 506免疫抑制剂药物复合物抑制。然而，有报道称FKB 51和其他FKBP亲免素能够独立于FK 506结合并抑制CN。FKBP 51在毛细血管内皮细胞（PMVEC）中表达，但在肺动脉内皮细胞（PAEC）中几乎不存在该蛋白水平。与PMVEC相比，PAEC表现出更高的SOC进入。Isoc通道在PAEC和PMVEC中的差异调节尚不完全清楚。FKBP 51可能在两种内皮细胞类型之间的这种差异调节中发挥作用。本项目验证了钙调神经磷酸酶调节TRPC 4在内皮Isoc通道上的去磷酸化状态，FKBP 51抑制钙调神经磷酸酶去磷酸化TRPC 4的能力，使通道活性降低的假设。 公共卫生相关性：内皮细胞由内衬血管的内皮细胞的半渗透层组成，当其被破坏时可导致某些病理，包括运动障碍和急性呼吸综合征。通过激活钙选择性Isoc电流，钙进入质膜导致内皮间隙形成，并显著促进内皮屏障破坏。Isoc通道尚未完全理解，因此更全面地辨别通道的分子组成，特别是参与通道活化的磷酸酶的身份/作用及其与大分子量亲免蛋白FKBP 51的可能的新型相互作用具有临床意义。

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 Unpublished results from our laboratory suggest that inactivation of the Isoc channel is dependent on phosphorylation of TRPC4.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. PUBLIC HEALTH RELEVANCE: Endothelium is comprised of a semi-permeable layer of endothelial cells lining blood vessels that when disrupted can lead to certain pathologies, including athlerosclerosis and acute respiratory syndrome. Calcium entry across the plasma membrane via activation of the calcium selective Isoc current leads to endothelial gap formation and contributes significantly to endothelial barrier disruption. The Isoc channel is not yet completely understood, and so it is of clinical relevance to more fully discern the molecular makeup of the channel, specifically the identity/role of the phosphatase involved in channel activation and its possible novel interactions with the large molecular weight immunophilin, FKBP51.