Regulation of V-ATPase-mediated Renal Proton Secretion

本申请者提出一项计划，为他将来从事研究肾脏的学术基础科学做准备。 功能，解决生理和病理条件下肾脏质子分泌的调节。 生理条件。这项研究将在丹尼斯·布朗博士的实验室进行 麻省理工学院膜生物学和肾脏单位项目。肾H+分泌主要由空泡介导 质子泵ATPase(V-ATPase)，一种也能酸化某些细胞内细胞器的酶。然而， 当在质膜上表达时，就像在集合管A型插入细胞(1C)中一样，V-ATPase 调节跨上皮H+的分泌。H+分泌缺陷会导致远端肾小管酸中毒(DRTA)， 与人类的感觉神经性耳聋有关。DRTA是由编码基因突变引起的 V-ATPase的56 kDa B1亚基亚基异构体。我们假设另一个V-ATPase B亚基，B2 异构体可能在功能上替代肾脏H+分泌中的B1 有些情况下，因为我们现有的B1亚基敲除小鼠不是酸性的，它们表达更多 B_2亚基在1C组小鼠顶膜中的表达高于正常组。了解B1和B2的方式 组装成V-ATPase复合体及其亚基在V-ATPase靶向和转运中的作用 这些过程可能会提出新的治疗策略，通过在细胞中进行“异构体替代疗法”，其中B1- 介导的H+分泌是有缺陷的。这项提议和培训计划的主要目标是使用新的 确定V-ATPase B1和B2亚型在H+中相对作用的技术和动物模型 肾上皮细胞的分泌和V-ATPase转运。B2膜是如何表达的 缺乏B1的小鼠的亚基增加，这一表达/贩运过程是如何调节的？我们会 B_1基因敲除小鼠脑内V-ATPase mRNA和蛋白表达的调节 不同的酸碱条件。用新的激光捕获显微切割技术从1C中分离出mRNA 在1C中表达EGFP的转基因小鼠将在1C中检测代偿性B2-V-ATPase功能 PH比值成像。B1相互作用蛋白NHE-RF1在V-ATPase运输中的作用将被解决， 碳酸氢盐刺激的可溶性腺苷环化酶(SAC)在V- 包含B1和B2亚基的ATPase将使用多学科方法进行检查。

This applicant proposes a program to prepare him for a career in academic basic science studying renal function, addressing the regulation of proton secretion in the kidney under physiological and patho- physiological conditions. The research will be conducted in the laboratory of Dr. Dennis Brown in the Program in Membrane Biology and Renal Unit, MGH. Renal H+ secretion is mainly mediated by the vacuolar proton-pumping ATPase (V-ATPase), an enzyme that also acidifies some intracellular organelles. However, when expressed on the plasma membrane, as in collecting duct A-type intercalated cells (1C),the V-ATPase mediates transepithelial H+ secretion. Defects in H+ secretion cause distal renal tubular acidosis (dRTA), associated with sensorineural deafness in humans. dRTA is caused by mutations in the gene encoding the 56 kDa B1 subunit isoform of the V-ATPase. We hypothesize that an alternative V-ATPase B subunit, the B2 isoform might serve as a replacement back-up that functionally replaces the B1 in renal H+ secretion under some conditions, because our available B1 subunit knockout mice are not acidotic and they express more B2 subunit in the apical membrane of 1C than do normal mice. Understanding the ways in which B1 and B2 assemble into V-ATPase complexes and the role of these subunits in V-ATPase targeting and trafficking processes could suggest novel treatment strategies by "isoform replacement therapy" in cells in which B1- mediated H+ secretion is defective. The main goal of this proposal and training program is use novel techniques and animal models to determine the relative role of the V-ATPase B1 and B2 isoforms in H+ secretion and V-ATPase trafficking in renal epithelial cells. How does the membrane expression of the B2 subunit increase in mice lacking B1, and how is this expression/trafficking process regulated? We will examine regulation of V-ATPase mRNA and protein expression in 1C from unique B1-knockout mice under different acid-base conditions. mRNA from 1C will be isolated by laser capture microdissection using new transgenic mice that express EGFP in 1C.Compensatory B2-V-ATPase function in 1C will be examined by pH ratio imaging. The role of the B1-interacting protein NHE-RF1 in V-ATPase trafficking will be addressed, and a role for the bicarbonate-stimulated soluble adenylate cyclase (sAC) in the membrane insertion of V- ATPases containing the B1 and B2 subunit will be examined using a multidisciplinary approach.