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，我的实验室在肾脏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一个关键的紧密连接蛋白复合体，它由三种claudin蛋白组成：claudin-14、claudin-16和claudin-19。人类claudin-16和claudin-19基因突变导致遗传性疾病FHHNC(家族性低镁血症伴高钙尿和肾钙质沉着症)。根据全基因组关联标准，人类claudin-14中的SNP与肾结石疾病有关。这项题为“Claudin-16和Claudin-19通道的生化和生物物理性质”的提案将检验这一中心假设，即通过Claudin的表达、功能、相互作用和结合而改变的细胞旁通透性是CaSR和PTH调节肾脏钙和镁重吸收的关键信号功能的基础。这项提议有三个目标。目的1阐明Claudin-14、-16和-19在肾脏中作用的生化和生物物理基础。我们将使用一系列生化和生物物理方法，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首次使我们能够在亚微米分辨率下揭示细胞旁的渗透性。目的2将阐明Claudin对CaSR和PTH信号的调节基础。我们将建立两个关键的小鼠模型-TALH特异性CaSR或PTH1R 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/PTH1R KO或公认的药理学方法研究CaSR和PTH信号在肾脏中的相互作用。目的3将确定Claudin在肾钙处理中的机制作用。Claudin单体或寡聚体的肾脏转运功能将基于我们建立的Claudin-14、-16、-19单一、双重和三重KO和Claudin-14过表达小鼠模型，使用组合基因敲除方法来确定。我们还将研究PTH、CaSR和microRNA如何基于Claudin表达、功能、相互作用和结合的变化，利用单肾单位灌流技术改变细胞旁通透性。

 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.