Chloride Channels in Lung Development

肺部发育中的氯离子通道

• 批准号: 7824125
• 负责人: Pamela L. Zeitlin
• 金额: $ 6.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7824125
• 关键词: Agonist; Alleles; Amiloride; Amino Acid Sequence; Animal Model; Apical; Aspirate substance; Bacteria; Binding; Birth; Bypass; CLC-2 protein; Cell membrane; Chloride Channels; Chloride Ion; Chlorides; Complex; Cyclic AMP; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Defect; Down-Regulation; Doxycycline; Epithelial; Epithelial Cells; Epithelial Physiology; Family health status; Financial compensation; Fluid Balance; Funding; Gel; Gene Expression; Genes; Genetic; Goals; Grant; Healthcare Systems; Heat-Shock Proteins 90; Human; Infant; Infection; Inflammation; Inflammatory; Inflammatory Response; Inherited; Ion Transport; Knock-out; Knockout Mice; Lead; Life; Liquid substance; Lung; Lung diseases; Mediating; Membrane Potentials; Methods; Modeling; Morbidity - disease rate; Mucociliary Clearance; Mus; Nanotechnology; Neutrophil Infiltration; Pathway interactions; Peptide Sequence Determination; Pharmacologic Substance; Proteins; Proteomics; Pseudomonas aeruginosa; Pulmonary Cystic Fibrosis; Respiratory physiology; Sodium; Sodium Channel; Surface; Techniques; Technology; Testing; Tetanus Helper Peptide; Transfection; Transgenic Mice; Transgenic Organisms; Trypsin; Viscosity; Work; absorption; airway epithelium; airway inflammation; apical membrane; cystic fibrosis mouse; cytokine; epithelial Na+ channel; fetal; glycosylation; in vivo; killings; lung development; mortality; mouse model; mutant; novel; novel therapeutic intervention; postnatal; protein protein interaction; public health relevance; trafficking; transcription factor; voltage

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.

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，会带来巨大的经济负担。持续进行性肺部疾病仍然是 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，此时它可以维持纤毛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 介导的氯离子分泌的情况下，上皮钠通道 ENaC 不受调节，导致钠和液体吸收过多。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。我们提出可以利用替代氯离子通道来绕过 CFTR 缺陷。我们将测试以下假设：出生后 ClC-2 通道的过度表达和激活将通过恢复氯离子分泌和 EN​​aC 下调来拯救 CF 小鼠肺。我们的长期目标是发现技术并确定允许用内源基因产物 ClC-2 补偿突变 CFTR 的机制。目标 1：确定 ClC2 靶向质膜的蛋白质相互作用序列。假设 HSP90 复合物调节 ClC-2 的顶端质膜表达。我们证明了 HSP90 与 ClC-2 相互作用，并且我们开发了在 2D 凝胶上识别 ClC-2 及其结合伙伴的技术，然后进行 MALDI-TOF。目标是发现最大化表面 ClC-2 表达和功能的因素。目标 2：确定 ClC-2 和 ENaC 是否通过 Cl-电流相互作用来调节 CF 小鼠肺部的离子转运。假设通过 ClC-2 的氯电导会抑制 ENaC 功能。我们预测，在 CF 小鼠中，ClC-2 的持续激活将降低静息电位差和阿米洛利抑制的 PD 部分。目标 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 小鼠炎症反应的正常水平。这些研究将产生以下新颖的贡献：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​​和共享新型上皮特异性、多西环素调节的 TET-On 人类 ClC- 2个小鼠模型； 3) 对 Sucampo Pharmaceuticals 的两种新型 ClC-2 激活剂治疗 CF 肺部疾病的评估； 4) 诱导和评估 CF 小鼠中 CFTR 介导的气道炎症的技术。公共健康相关性：我们建议检验以下假设：pH 和电压激活的 ClC-2 通道的过度表达和激活将拯救囊性纤维化小鼠。关键蛋白质：在 ClC-2 运输到气道上皮细胞顶膜的过程中，蛋白质相互作用将受到质疑。这项工作的产品将是一种刺激氯离子转运的方法，以补偿突变的 CFTR、减少钠吸收并缓解体内气道炎症。