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: 8450188
• 负责人: Jianghui Hou
• 金额: $ 30.13万
• 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/8450188
• 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(申请人提供)：肾脏离子和溶质细胞旁渗透的生理学是非常重要的，但人们对此知之甚少。克劳丁是细胞旁途径的关键成分。克拉丁功能缺陷会导致多种肾脏疾病，包括低镁血症、高钙尿、低氯血症和盐敏性高血压。人类claudin-16和claudin-19基因突变导致遗传性肾脏疾病FHHNC(家族性低镁血症伴高钙尿和肾脏钙质沉着症)。我们发现，这两个Claudin共定位在粗大的升支(TAL)，相互作用，形成阳离子选择性蛋白复合体。我们已经建立了claudin-16基因敲除(KD)和claudin-19kD小鼠品系。Claudin-16KD动物表现出慢性肾脏镁和钙的消耗，发展成与人类FHHNC患者相似的肾脏钙质沉着症。这项研究将研究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镁离子的集体作用。具体目标1旨在了解claudin-16和claudin-19如何共同寡聚并组装成紧密连接的链。这些研究将确定claudin-16/claudin-19寡聚复合体的化学计量比，并通过代谢追逐分析确定claudin寡聚的关键细胞内步骤。具体目的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的聚乙二醇寡聚体(半径2.8-7)的胞外通量，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关键氨基酸位点。这些研究将测试两种claudin-16和claudin-19通道的结构和功能：(1)claudin-16和claudin-19形成两个平行的同质通道，每个通道都有自己的生理特征；(2)claudin-16和claudin-19形成具有新特性的异构体通道，需要它们的协同作用。具体目标3包括对claudin-16kD小鼠、claudin-19kD(KO)小鼠和claudin-16kD+claudin-19kD(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，并使用单管灌流技术记录这些小鼠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在体内正常发挥功能所必需的。