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

描述(申请人提供)：内皮细胞排列血管，并允许分子的选择性运输。破坏这一屏障会导致渗透性增加，从而 可导致某些病理，包括炎症、急性呼吸综合征和运动性硬化症。细胞内钙离子通过钙库操作的钙通道(SOC)进入，对屏障的破坏有重要作用。Isoc是一种钙选择性SOC电流，它的激活导致内皮细胞缝隙的形成。瞬时受体典型(TRPC)蛋白TRPC1和TRPC4是Isoc通道结构的一部分，TRPC4是Isoc通道正常功能所必需的。当Isoc被激活时，该通道打开，导致钙跨细胞膜内流。相反，通道关闭会限制钙离子的进入。最近的研究表明，TRPC3通道丝氨酸712的磷酸化是导致该通道负调控的原因。我们实验室未发表的结果表明，Isoc通道的失活依赖于TRPC4的磷酸化。似乎TRPC4在内部钙储存被释放时被去磷酸化，从而允许通道激活，并在Isoc通道开放后被磷酸化。了解参与通道激活准备的磷酸酶对于Isoc通道调节的更大图景至关重要。磷酸酶调节TRPC4的磷酸化状态的特性和作用尚不清楚。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被免疫亲和素FKBP51-FK506免疫抑制药物复合体所抑制。然而，已经有报道表明FKB51和其他FKBP免疫亲和素能够独立于FK506与CN结合并抑制CN。FKBP51在毛细血管内皮细胞(PMVECs)中表达，但在肺动脉内皮细胞(PAECs)中几乎不表达。与PMVEC相比，PAEC表现出更高的SOC条目。PAECs和PMVECs对Isoc通道的不同调控尚不完全清楚。FKBP51可能在这两种内皮细胞类型之间的这种差异调节中发挥作用。本项目验证了一种假设，即钙调神经磷酸酶调节内皮细胞Isoc通道上TRPC4的去磷酸化状态，而FKBP51抑制钙调神经磷酸酶去磷酸化TRPC4的能力，使该通道不那么活跃。 与公共卫生相关：内皮细胞是由血管内皮细胞组成的一层半透层，当内皮细胞被破坏时，可能会导致某些病理变化，包括动脉粥样硬化和急性呼吸综合征。钙离子通过激活钙离子选择性等离子电流进入质膜，导致内皮细胞间隙的形成，并对内皮屏障的破坏起重要作用。Isoc通道尚不完全清楚，因此更充分地了解该通道的分子组成具有临床意义，特别是参与通道激活的磷酸酶的身份/作用及其可能与大分子免疫亲和素FKBP51的新的相互作用。

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 Unpublished results from our laboratory suggest that inactivation of the Isoc channel is dependent on phosphorylation of TRPC4.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.