BIOCHEMICAL AND BIOPHYSICAL NATURE OF CLAUDIN-16 AND CLAUDIN-19 CHANNELS

Claudin-16 和 Claudin-19 通道的生物化学和生物物理性质

• 批准号: 8247835
• 负责人: Jianghui Hou
• 金额: $ 31.22万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8247835
• 关键词: Address; Alkalies; Amino Acids; Animals; Anions; Back; Basic Science; Binding; Biochemical; Biological; Biology; Blood; Calcium; Cations; Cell physiology; Cells; Cellular biology; Charge; Chemicals; Chronic; Clinical; Complex; Cysteine; Data; Defect; Detergents; Divalent Cations; Dominant-Negative Mutation; Epithelial; Epithelial Cells; Failure; Family; Grant; Human; Hypertension; Hypomagnesemia; Inherited; Intercellular Junctions; Ions; Kidney; Kidney Diseases; Knockout Mice; Limb structure; Magnesium; Measurement; Measures; Metabolic; Modeling; Molecular Biology; Mus; Mutagenesis; Mutation; Nature; Nephrocalcinosis; Nephrons; Operative Surgical Procedures; Pathway interactions; Patients; Perfusion; Physiological; Physiology; Property; Public Health; Radial; Renal clearance function; Renal function; Role; Running; Salts; Scanning; Series; Side; Site-Directed Mutagenesis; Sodium Chloride; Structure; Sucrose; System; Techniques; Testing; Thick; Tight Junctions; Urine; claudin-1 protein; crosslink; design; hypercalciuria; hypochloremia; in vivo; member; mouse model; novel; protein complex; public health relevance; research study; salt sensitive; sedimentation velocity; small molecule; solute; stoichiometry; wasting

DESCRIPTION (provided by applicant): The physiology of paracellular permeation of ions and solutes in the kidney is pivotally important but poorly understood. Claudins are the key components of the paracellular pathway. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria, hypochloremia and salt- sensitive hypertension. Human mutations in claudin-16 and claudin-19 are responsible for the hereditary renal disease FHHNC (Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis). We have found that the two claudins co-localize in the thick ascending limb (TAL), interact and form a cation-selective protein complex. We have developed claudin-16 knockdown (KD) and claudin-19 KD mouse lines. Claudin-16 KD animals show chronic renal wasting of magnesium and calcium, developing renal nephrocalcinosis comparable to that seen in human patients with FHHNC. This proposal will study the molecular interaction between claudin-16 and claudin-19, the size and charge selectivity of claudin-16 and claudin-19 channel pore and their collective effects on renal handling of Mg++. Specific Aim 1 intends to understand how claudin-16 and claudin-19 co-oligomerize and assemble into the tight junction strands. These studies will determine the stoichiometry of claudin-16/claudin- 19 oligomeric complex and identify critical intracellular step in claudin oligomerization using metabolic chase analyses. Specific Aim 2 intends to elucidate the biophysical properties of the claudin-16 and claudin-19 channels. These studies will measure the paracellular flux of a continuous series of PEG oligomers (of radius 2.8 - 7 ¿) to unveil the size selectivity of claudin-16 and claudin-19 channels. These studies will identify key loci of amino acids in claudin-16 and claudin-19 required for their charge selectivity. These studies will test two models of claudin-16 and claudin-19 channel structure and function: (1) claudin-16 and claudin-19 form two parallel homomeric channels each with its own physiologic signature; (2) claudin-16 and claudin-19 form a heteromeric channel with novel properties that require their synergy. Specific Aim 3 involves careful phenotypic analyses of claudin-16 KD mice, claudin-19 KD (KO) mice and claudin-16 KD + claudin-19 KD (KO) mice. These studies will analyze the renal clearance and transport functions in these mice, and record the electrophysiological properties of claudin-16 and claudin-19 channels in the TAL of these mice using single tubule perfusion techniques. These studies intend to test if the interaction between claudin-16 and claudin-19 is required for normal function of the TAL in vivo. PUBLIC HEALTH RELEVANCE: Kidneys function by initially excreting many salts and small molecules found in the blood, then selectively taking back those that need to be conserved while allowing others to be excreted in the urine. This grant will study claudin functions of cell-cell junctions that provide one of the key pathways (the paracellular pathway) used by the kidney to move salt between urine and blood. Defects in claudin function result in a broad range of renal diseases, including hypomagnesemia, hypercalciuria, hypochloremia and salt- sensitive hypertension.

描述（由申请人提供）：肾脏中离子和溶质的细胞旁渗透的生理学至关重要，但人们对此知之甚少。密蛋白是细胞旁途径的关键组成部分。密蛋白功能缺陷会导致多种肾脏疾病，包括低镁血症、高钙尿症、低氯血症和盐敏感性高血压。人类claudin-16和claudin-19突变导致遗传性肾病FHHNC（家族性低镁血症伴高钙尿症和肾钙质沉着症）。我们发现这两个紧密蛋白共定位于粗升肢（TAL），相互作用并形成阳离子选择性蛋白复合物。我们开发了claudin-16敲低（KD）和claudin-19 KD小鼠品系。 Claudin-16 KD 动物表现出镁和钙的慢性肾脏消耗，发展为肾钙质沉着症，与人类 FHHNC 患者中所见的情况相当。该提案将研究claudin-16和claudin-19之间的分子相互作用、claudin-16和claudin-19通道孔的大小和电荷选择性以及它们对肾脏处理Mg++的集体影响。具体目标 1 旨在了解claudin-16 和claudin-19 如何共寡聚并组装成紧密连接链。这些研究将确定claudin-16/claudin-19寡聚复合物的化学计量，并使用代谢追踪分析确定claudin寡聚化中的关键细胞内步骤。具体目标 2 旨在阐明claudin-16 和claudin-19 通道的生物物理特性。这些研究将测量一系列连续 PEG 寡聚物（半径 2.8 - 7 ¿）的细胞旁通量，以揭示 claudin-16 和 claudin-19 通道的尺寸选择性。这些研究将确定claudin-16 和claudin-19 中电荷选择性所需的关键氨基酸位点。这些研究将测试claudin-16和claudin-19通道结构和功能的两种模型：（1）claudin-16和claudin-19形成两个平行的同聚通道，每个通道都有自己的生理特征； (2)claudin-16和claudin-19形成异聚通道，其具有需要它们协同作用的新特性。具体目标 3 涉及对claudin-16 KD 小鼠、claudin-19 KD (KO) 小鼠和claudin-16 KD + claudin-19 KD (KO) 小鼠进行仔细的表型分析。这些研究将分析这些小鼠的肾脏清除和转运功能，并使用单管灌注技术记录这些小鼠TAL中claudin-16和claudin-19通道的电生理特性。这些研究旨在测试claudin-16 和claudin-19 之间的相互作用是否是TAL 体内正常功能所必需的。 公共健康相关性：肾脏的功能首先是排出血液中的许多盐和小分子，然后选择性地收回那些需要保留的盐，同时允许其他盐和小分子从尿液中排出。这笔赠款将研究细胞与细胞连接的密蛋白功能，该连接提供了肾脏用于在尿液和血液之间移动盐的关键途径之一（细胞旁途径）。密蛋白功能缺陷会导致多种肾脏疾病，包括低镁血症、高钙尿症、低氯血症和盐敏感性高血压。