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

 描述（由申请人提供）：在过去的几年里，我的实验室在肾钙和镁处理的细胞旁通道方面取得了关键发现。我们故事的中心是来自肾脏的一种关键的紧密连接蛋白复合物，它由三种claudin蛋白组成：claudin-14，claudin-16和claudin-19。人的claudin-16和claudin-19突变导致遗传性疾病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“密封蛋白-16和密封蛋白-19通道的生物化学和生物物理性质”的提案将检验中心假设，即通过密封蛋白表达、功能、相互作用和组合的细胞旁渗透性的变化是CaSR和PTH调节肾钙和镁重吸收的关键信号功能的基础。这项建议有三个目的。目的1将阐明claudin-14、-16和-19在肾脏中功能的生化和生物物理基础。我们将使用一系列的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）建立的一种新的记录方法。SICM首次使我们能够以亚微米分辨率揭示细胞旁渗透性。目的2将阐明claudin对CaSR和PTH信号的调节基础。我们将产生两个关键的小鼠模型- TALH特异性CaSR或PTH 1 R KO小鼠，以破译CaSR和PTH对claudins的特异性调节。我们还将使用双CaSR/PTH 1 R KO或成熟的药理学方法研究CaSR和PTH信号如何在肾脏中相互作用。目的3将确定密蛋白在肾钙处理中的机制作用。基于我们已经建立的封闭蛋白KO小鼠模型-封闭蛋白-14、-16、-19单、双和三重KO和封闭蛋白-14过表达小鼠，使用组合敲除方法测定封闭蛋白单体或寡聚体的肾转运功能。我们还将使用单肾单位灌注技术研究PTH、CaSR和microRNA如何根据密蛋白表达、功能、相互作用和组合的变化改变细胞旁渗透性。

 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.