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: 9064757
• 负责人: Jianghui Hou
• 金额: $ 32.54万
• 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 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9064757
• 关键词: Affect; Agonist; Animal Model; Animals; Architecture; Back; Biochemical; Blood; CLDN14 gene; Calcium; Cell model; Cell physiology; Cells; Defect; Detergents; Disease; Excretory function; Gene Expression; Genes; Grant; Health; Hereditary Disease; Homeostasis; Human; Hypomagnesemia; Insecta; Intercellular Junctions; Kidney; Kidney Calculi; Kidney Diseases; Knock-out; Knockout Mice; Knowledge; LoxP-flanked allele; Magnesium; Mediating; MicroRNAs; Molecular; Molecular Weight; Mus; Mutation; Nature; Nephrocalcinosis; Nephrons; Parathyroid gland; Pathway interactions; Perfusion; Permeability; Physiological; Physiology; Property; Proteins; Publications; Regulation; Renal function; Resolution; Role; Salts; Sedimentation process; Series; Signal Pathway; Signal Transduction; Sodium Chloride; Solubility; Structure of ascending limb of Henle' s loop; Structure-Activity Relationship; Techniques; Testing; Thick; Tight Junctions; Time; Transgenic Animals; Transgenic Organisms; Urine; Work; base; biophysical techniques; combinatorial; extracellular; genome wide association study; hypercalciuria; monomer; mouse model; novel; overexpression; protein complex; reconstitution; response; scanning ion conductance microscopy; small molecule; stoichiometry; submicron; urinary

 DESCRIPTION (provided by applicant): For the past several years, my lab has made key discoveries of the paracellular channel underlying renal calcium and magnesium handling. At the center of our story is a key tight junction protein complex from the kidney that comprises of three claudin proteins: claudin-14, claudin-16 and claudin-19. Human mutations in claudin-16 and claudin-19 cause the hereditary disease FHHNC (Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis). Human SNPs in claudin-14 are associated with kidney stone diseases according to genome-wide association criteria. This proposal entitled "Biochemical and biophysical nature of claudin-16 and claudin-19 channels" will test the central hypothesis that the changes in paracellular permeabilities through claudin expression, function, interaction and combination underlie the key signaling functions of CaSR and PTH to regulate renal calcium and magnesium reabsorption. There are three aims in this proposal. Aim 1 will elucidate the biochemical and biophysical basis of claudin-14, -16 and -19 functions in the kidney. We will use a range of biochemical and biophysical approaches with emphasis on a novel recording approach established by our lab based upon Scanning Ion Conductance Microscopy (SICM). SICM has, for the first time, allowed us to reveal paracellular permeabilities at submicron resolution. Aim 2 will elucidate the regulatory basis for claudin in response to CaSR and PTH signaling. We will generate two key mouse models - TALH specific CaSR or PTH1R KO mice to decipher the CaSR and PTH specific regulation of claudins. We will also study how CaSR and PTH signaling cross talk in the kidney using double CaSR/PTH1R KO or a well-established pharmacological approach. Aim 3 will determine the mechanistic role of claudin in renal calcium handling. The renal transport function of claudin monomer or oligomer will be determined using combinatorial knockout approaches based upon the claudin KO mouse models we have established - claudin-14, -16, -19 single, double and triple KO and claudin- 14 overexpression mice. We will also study how PTH, CaSR and microRNA alter paracellular permeabilities based upon changes in claudin expression, function, interaction, and combination using single nephron perfusion techniques.

 描述（由申请人提供）：在过去的几年中，我的实验室在肾钙和镁处理的细胞旁通道方面取得了重要发现。我们故事的中心是来自肾脏的关键紧密连接蛋白复合物，它由三种紧密连接蛋白组成：claudin-14、claudin-16 和claudin-19。人类claudin-16和claudin-19突变会导致遗传性疾病FHHNC（家族性低镁血症伴高钙尿症和肾钙质沉着症）。根据全基因组关联标准，claudin-14 中的人类 SNP 与肾结石疾病相关。这项题为“claudin-16 和 claudin-19 通道的生物化学和生物物理性质”的提案将检验一个中心假设，即通过claudin 表达、功能、相互作用和组合引起的细胞旁通透性的变化是 CaSR 和 PTH 调节肾钙和镁重吸收的关键信号传导功能的基础。该提案有三个目标。目标 1 将阐明claudin-14、-16 和-19 在肾脏中的功能的生化和生物物理基础。我们将使用一系列生物化学和生物物理方法，重点是我们实验室基于扫描离子电导显微镜 (SICM) 建立的新颖记录方法。 SICM 首次使我们能够以亚微米分辨率揭示细胞旁通透性。目标 2 将阐明密蛋白响应 CaSR 和 PTH 信号传导的调控基础。我们将生成两个关键小鼠模型 - TALH 特异性 CaSR 或 PTH1R KO 小鼠，以破译密蛋白的 CaSR 和 PTH 特异性调节。我们还将研究 CaSR 和 PTH 信号如何在肾脏中使用双 CaSR/PTH1R KO 或成熟的药理学方法进行交互。目标 3 将确定紧密蛋白在肾钙处理中的机制作用。密蛋白单体或寡聚体的肾转运功能将使用基于我们建立的密蛋白KO小鼠模型（claudin-14、-16、-19单、双和三重KO以及claudin-14过表达小鼠）的组合敲除方法来确定。我们还将研究 PTH、CaSR 和 microRNA 如何基于 Claudin 表达、功能、相互作用和组合的变化，使用单肾单位灌注技术来改变细胞旁通透性。