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: 8587125
• 负责人: Jianghui Hou
• 金额: $ 0.15万
• 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/8587125
• 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; 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.

描述（由申请人提供）：肾脏中离子和溶质的细胞旁渗透生理学非常重要，但了解甚少。紧密连接蛋白是细胞旁途径的关键组分。紧密连接蛋白功能的缺陷导致广泛的肾脏疾病，包括低镁血症、高钙尿、低镁血症和盐敏感性高血压。人的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）。我们已经发现，这两个claudins共定位在厚上升肢（TAL），相互作用，形成一个阳离子选择性蛋白复合物。我们已经开发了claudin-16敲除（KD）和claudin-19 KD小鼠系。紧密连接蛋白-16 KD动物显示镁和钙的慢性肾消耗，发展成与患有FHHNC的人类患者中所见相当的肾性肾钙质沉着症。本研究将研究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旨在了解紧密连接蛋白-16和紧密连接蛋白-19如何共寡聚并组装成紧密连接链。这些研究将确定密蛋白-16/密蛋白-19寡聚复合物的化学计量，并使用代谢追踪分析鉴定密蛋白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。这些研究将测量连续系列的PEG低聚物（半径为2.8 - 7 μ m）的细胞旁通量，以揭示密蛋白-16和密蛋白-19通道的尺寸选择性。这些研究将确定其电荷选择性所需的密蛋白-16和密蛋白-19中的关键氨基酸位点。这些研究将测试紧密连接蛋白-16和紧密连接蛋白-19通道结构和功能的两种模型：（1）紧密连接蛋白-16和紧密连接蛋白-19形成两个平行的同聚体通道，每个通道具有其自身的生理特征;（2）紧密连接蛋白-16和紧密连接蛋白-19形成具有需要其协同作用的新特性的异聚体通道。具体目标3涉及紧密连接蛋白-16 KD小鼠、紧密连接蛋白-19 KD（KO）小鼠和紧密连接蛋白-16 KD +紧密连接蛋白-19 KD（KO）小鼠的仔细表型分析。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。这些研究旨在测试密封蛋白-16和密封蛋白-19之间的相互作用是否是TAL体内正常功能所需的。