Regulation of V-ATPase-mediated Renal Proton Secretion

描述(由申请人提供)： 这位申请者提出了一个计划，为他从事学术基础科学研究肾脏功能做准备，研究生理和病理生理条件下肾脏质子分泌的调节。这项研究将在麻省理工学院膜生物学和肾脏单位项目的丹尼斯·布朗博士的实验室进行。肾脏H的分泌主要由液泡质子泵ATPase(V-ATPase)调节，该酶还能酸化某些细胞内的细胞器。然而，当V-ATPase表达在质膜上时，就像在集合管A型间质细胞(IC)中一样，V-ATPase介导了跨上皮H的分泌。H分泌缺陷导致远端肾小管性酸中毒(DRTA)，与人类感音神经性耳聋相关。DRTA是由编码V-ATPase的56 kDa B1亚基异构体的基因突变引起的。我们推测，另一种V-ATPase B亚基B2亚型在某些条件下可能在功能上替代肾H分泌中的B1，因为我们现有的B1亚基敲除小鼠不是酸性的，而且它们在IC的顶膜上比正常小鼠表达更多的B2亚基。了解B1和B2组装成V-ATPase复合体的方式以及这些亚单位在V-ATPase靶向和运输过程中的作用，可以为B1介导的H分泌缺陷的细胞提供新的治疗策略。这项建议和培训计划的主要目标是使用新的技术和动物模型来确定V-ATPase B1和B2亚型在肾上皮细胞H分泌和V-ATPase转运中的相对作用。在缺乏B1的小鼠中，B2亚基的膜表达是如何增加的，这个表达/运输过程是如何调节的？我们将研究在不同的酸碱条件下，唯一的B1基因敲除小鼠的IC中V-ATPase的mRNA和蛋白表达的调节。利用激光捕获显微切割技术，利用在IC中表达绿色荧光蛋白的新型转基因小鼠，从IC中分离出mRNA。用pH比值显像法检测IC的代偿性B2-V-ATPase功能。将讨论B1相互作用蛋白NHE-RF1在V-ATPase运输中的作用，并将使用多学科方法研究重碳酸盐刺激的可溶性腺苷酸环酶(SAC)在含有B1和B2亚基的V-ATPase膜插入中的作用。

DESCRIPTION (provided by applicant): 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 (IC), 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 IC 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 IC from unique B1-knockout mice under different acid-base conditions. mRNA from IC will be isolated by laser capture microdissection using new transgenic mice that express EGFP in IC. Compensatory B2-V-ATPase function in IC 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.