Chloride Channels in Lung Development

描述（由申请人提供）：囊性纤维化（CF）是一种限制生命的系统性遗传性疾病，具有巨大的经济负担。慢性进行性肺部疾病仍是CF发病和死亡的主要原因。囊性纤维化跨膜调节因子（CFTR）是一种cAMP调节的氯离子通道，在胎儿气道上皮细胞中高度表达。然而，直到出生后才需要CFTR功能，此时它维持睫状体周围液体平衡并维持有效的粘膜纤毛清除。出生后，在缺乏CFTR介导的氯分泌的情况下，上皮钠通道ENaC不受调节，并驱动过量的钠和液体吸收。这会使气道脱水并增加气道分泌物的粘度，从而损害细菌清除。我们已经提出，可以利用替代氯离子通道来绕过CFTR缺陷。我们将测试这一假设，即在出生后的C1 C-2通道的过度表达和激活将通过恢复氯分泌和下调ENaC来拯救CF小鼠肺。我们的长期目标是发现技术并确定机制，该机制将允许用内源性基因产物ClC-2补偿突变CFTR。目的1：确定ClC 2靶向质膜的蛋白质相互作用序列。假设HSP 90复合物调节ClC-2的顶端质膜表达。我们证明了HSP 90与ClC-2相互作用，并且我们已经开发了在二维凝胶上识别ClC-2及其结合伴侣的技术，然后进行MALDI-TOF。目的是发现使表面ClC-2表达和功能最大化的因素。目的2：确定ClC-2和ENaC是否通过Cl-电流相互作用来调节CF小鼠肺中的离子转运。假设是通过ClC-2的氯离子电导将抑制ENaC功能。我们预测，在CF小鼠中，持续激活ClC-2将降低静息电位差和PD的阿米洛利抑制分数。目的3：确定ClC-2功能是否可以调节对感染或LPS攻击的炎症反应。假设刺激ClC-2表达和ClC-2介导的氯离子转运将恢复CF小鼠中正常水平的炎症反应。这些研究将产生以下新的贡献：1）证明替代氯离子通道可以绕过CF动物模型中的CFTR缺陷：2）传播和共享新的上皮特异性的、多西环素调节的TET-On人ClC-2小鼠模型; 3）评估来自Sucampo Pharmaceuticals的两种新的ClC-2激活剂用于CF肺病;和4）在CF小鼠中诱导和评估CFTR介导的气道炎症的技术。公共卫生相关性：我们建议测试的假设，过度表达和激活的pH和电压激活的ClC-2通道将拯救囊性纤维化小鼠。关键蛋白质：蛋白质相互作用将在运输的ClC-2的气道上皮细胞的顶端膜进行查询。这项工作的结果将是一种刺激氯离子转运的方法，以补偿突变的CFTR，减少钠吸收和减轻体内气道炎症。

DESCRIPTION (provided by applicant): Cystic Fibrosis (CF) is a life-limiting systemic inherited illness with a tremendous financial burden. Relentlessly progressive pulmonary disease is still the primary cause of the morbidity and mortality in CF. The cystic fibrosis transmembrane regulator (CFTR) is a cAMP-regulated chloride channel that is highly expressed in fetal airway epithelia. However, CFTR function is not required until after birth when it maintains periciliary fluid balance and sustains effective mucociliary clearance. After birth, and in the absence of CFTR-mediated chloride secretion, the epithelial sodium channel, ENaC, is unregulated and drives excessive sodium and fluid absorption. This dehydrates the airways and increases the viscosity of airways secretions, thereby impairing bacterial clearance. We have proposed that alternative chloride channels can be exploited to bypass the CFTR defect. We will test the hypothesis that over-expression and activation of the ClC-2 channel in the postnatal period will rescue the CF murine lung by restoring chloride secretion and down-regulation of ENaC. Our long term goal is to discover the technology and determine the mechanism that will allow compensation of the mutant CFTR with an endogenous gene product, ClC-2. Aim 1: To determine the sequence of protein interactions by which ClC2 is targeted to the plasma membrane. The hypothesis is that HSP90 complexes regulate apical plasma membrane expression of ClC-2. We demonstrate that HSP90 interacts with ClC-2 and we have developed the technology to identify ClC-2 and its binding partners on 2D gels followed by MALDI-TOF. The goal is to discover the factors that maximize surface ClC-2 expression and function. Aim 2: To determine whether ClC-2 and ENaC interact through Cl- current to modulate ion transport in the CF mouse lung. The hypothesis is that chloride conductance through ClC-2 will inhibit ENaC function. We predict that in CF mice, sustained activation of ClC-2 will decrease resting potential difference and the amiloride inhibited fraction of PD. Aim 3: To determine whether ClC-2 function can regulate the inflammatory response to infection or LPS challenge. The hypothesis is that stimulating ClC-2 expression and ClC-2 mediated chloride transport will restore normal levels of inflammatory responses in CF mice. These studies will produce the following novel contributions: 1) demonstration that an alternative chloride channel can bypass the CFTR defect in an animal model of CF: 2) dissemination and sharing of a novel epithelial-specific, doxycycline-regulated TET-On human ClC-2 mouse model; 3) assessment of two novel ClC-2 activators, from Sucampo Pharmaceuticals, for CF lung disease; and 4) techniques to induce and assess CFTR-mediated airways inflammation in CF mice. PUBLIC HEALTH RELEVANCE: We propose to test the hypothesis that over-expression and activation of pH- and voltage activated ClC-2 channels will rescue the cystic fibrosis mouse. Critical protein:protein interactions will be interrogated during trafficking of ClC-2 to apical membranes of airway epithelial cells. The product of this work will be a method of stimulating chloride transport to compensate for mutant CFTR, reduce sodium absorption and relieve airway inflammation in vivo.