ERP29: PROMOTING ION CHANNEL BIOGENESIS FROM THE ER LUMEN

项目总结/摘要 囊性纤维化（CF）和高血压是上皮细胞离子转运的缩短寿命的疾病。在CF 当囊性纤维化跨膜传导调节因子（CFTR）功能缺失时， 上皮钠通道（ENaC）活性的相对和不平衡增加，导致Na+ 呼吸道表面液体的吸收和消耗。反过来，这会损害粘膜纤毛清除， 使CF气道易于细菌定植/感染。ENaC也可以在导致 高血压;肾单位远端小管中ENaC功能异常增加导致Na+增加 尿潴留、血容量增加和高血压。因此，ENaC活性的调节是 了解和开发这些上皮离子转运疾病的治疗方法。 ENaC的开放概率（Po）以及因此其功能的关键决定因素是蛋白质的蛋白水解切割。 其α和γ亚基的腔/细胞外环;未切割的通道具有Po ~0，而完全切割的通道具有Po ~0， 有Po ~1。事实上，在CF气道上皮细胞中报道了增加的ENaC切割。有趣的是，ENaC可以 以切割或未切割的形式递送至上皮表面，但决定细胞增殖的因素 ENaC在生物发生过程中是否经历裂解尚不清楚。 我们已经广泛研究了4-苯基丁酸钠（4PBA），原型小分子校正剂， 导致CFTR突变的最常见CF的异常运输，ΔF508。其他研究表明，4PBA 还增加了ENaC的功能。我们发现ERp 29（Endoplasmic Reticulum Protein of 29 kD），一种新的， 广泛表达的内质网（ER）腔伴侣在细支气管中的表达增加， 在用4PBA处理的上皮细胞中，ERp 29促进野生型和ΔF508-CFTR的运输。 因此，ERp 29是第一个被证明积极影响CFTR运输的ER管腔组分。 我们最近也证明了ERp 29促进ENaC的功能。有趣的是，我们的数据 提示ERp 29通过在生物发生过程中指导ENaC在高尔基体中裂解来实现这一点， ERp 29降低ENaC功能，而不改变ENaC在顶端上皮表面的表达。的 该建议假设是上皮离子通道的腔面（如CFTR）和 ENaC，与ERp 29在他们的生物发生过程中是必需的，以指导这些客户端从ER到高尔基体。这 将通过研究对假设进行检验，具体目标是：1）检验是否存在CFTR的ER-管腔面对突变， 导致CF和预测不与ERp 29相互作用的人具有异常成熟。2)为了测试该机制， ERp 29在生物发生过程中将ENaC引导到高尔基体。3)为了测试ENaC能够绕过 在高尔基体中的分裂到达质膜。这些研究将产生关键的机制见解， 在设计CFTR和ENaC疾病的潜在ERp 29导向治疗干预中至关重要。

PROJECT SUMMARY/ABSTRACT Cystic Fibrosis (CF) and hypertension are life-shortening diseases of epithelial ion transport. In the CF airway, when the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is functionally absent, there is a relative and unbalanced increase in the activity of the Epithelial Sodium Channel (ENaC) that leads to Na+ absorption from and depletion of the airway surface liquid. In turn, this impairs mucociliary clearance and predisposes the CF airway to bacterial colonization/infection. ENaC can also play a key role in causing hypertension; abnormally increased function of ENaC in the distal tubule of the nephron leads to increased Na+ retention, increased blood volume and hypertension. The regulation of ENaC activity is therefore key in understanding and developing therapies for these diseases of epithelial ion transport. A critical determinant of ENaC's open probability (Po), and therefore its function, is the proteolytic cleavage of the luminal/extracellular loops of its α and γ subunits; uncleaved channel has Po ~0, while fully cleaved channel has Po ~1. In fact, increased ENaC cleavage is reported in CF airway epithelial cells. Interestingly, ENaC can be delivered to the epithelial surface in either a cleaved or an uncleaved form, but the factor(s) that determine whether or not ENaC undergoes cleavage during biogenesis are not known. We have extensively studied Sodium 4-Phenylbutyrate (4PBA), the prototype small molecule corrector of the aberrant trafficking of the most common CF causing CFTR mutation, ΔF508. Others demonstrated that 4PBA also increases the function of ENaC. We found that ERp29 (Endoplasmic Reticulum Protein of 29 kD), a novel, ubiquitously expressed endoplasmic reticulum (ER) luminal chaperone has increased expression in bronchiolar epithelial cells treated with 4PBA, and that ERp29 promotes the trafficking of both wild type and ΔF508-CFTR. ERp29 was thus the first ER luminal component demonstrated to positively influence CFTR trafficking. We have also recently demonstrated that ERp29 promotes the function of ENaC. Interestingly, our data suggest that ERp29 does this by directing ENaC for cleavage in the Golgi during biogenesis, as depletion of ERp29 decreased ENaC function without altering expression of ENaC at the apical epithelial surface. The hypothesis of this proposal is that interaction of the luminal face of epithelial ion channels, such as CFTR and ENaC, with ERp29 during their biogenesis is required to direct these clients from the ER to the Golgi. This hypothesis will be tested with studies Specifically Aiming: 1) To test if ER-luminal facing mutations of CFTR that cause CF and are predicted to not interact with ERp29 have abnormal maturation. 2) To test the mechanism by which ERp29 directs ENaC to the Golgi during biogenesis. 3) To test the mechanism by which ENaC can bypass cleavage in the Golgi en route to the plasma membrane. These studies will yield key mechanistic insights that will be crucial in designing potential ERp29-directed therapeutic interventions for CFTR- and ENaC-opathies.