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: 8053454
• 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/8053454
• 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）：肾中离子和溶质的细胞旁渗透的生理学是至关重要的，但知之甚少。claudin是细胞旁通路的关键成分。claudin功能缺陷导致广泛的肾脏疾病，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的突变是遗传性肾病FHHNC（家族性低镁血症伴高钙尿症和肾钙质沉着症）的原因。我们已经发现这两个cladin在厚升肢（TAL）中共定位，相互作用并形成阳离子选择性蛋白复合物。我们建立了claudin-16敲低（KD）和claudin-19 KD小鼠系。claudin - 16kd动物表现出慢性肾镁和钙的消耗，发生肾钙沉着症，与人类FHHNC患者相似。本课题将研究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及其对肾处理Mg++的集体影响。Specific Aim 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寡聚物的化学计量学，并利用代谢追踪分析确定claudin寡聚化的关键细胞内步骤。特异性目的2旨在阐明claudin-16和claudin-19通道的生物物理性质。这些研究将测量一系列连续的PEG低聚物（半径为2.8 - 7¿）的细胞旁通量，以揭示claudin-16和claudin-19通道的大小选择性。这些研究将确定claudin-16和claudin-19中电荷选择性所需的氨基酸的关键位点。这些研究将测试两种claudin-16和claudin-19通道结构和功能模型：(1)claudin-16和claudin-19形成两个平行的同源通道，每个通道都有自己的生理特征；(2) claudin-16和claudin-19形成了一个具有新特性的异质通道，需要它们的协同作用。特异性目的3包括对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。本研究将采用单小管灌注技术分析这些小鼠的肾脏清除率和转运功能，并记录这些小鼠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需要在体内TAL的正常功能。