An epithelial model for V-type H+-ATPase-driven acid-base transport

描述（由申请人提供）： 项目概述：本申请旨在将候选人（Peter Piermarini）从博士后科学家转变为独立研究员。促进这一转变是他的导师教授克劳斯Beyenbach和迈克尔Kotlikoff，和两个外部顾问（硼，耶鲁大学和哈里森，亚利桑那州）。在拟议的研究中，候选人将（i）扩展他的分子和基因组生物学实验库，（ii）获得肾生理学的强大方法，如肾小管上皮细胞的体外微灌注，以及（iii）获得电生理学的扎实知识。除了研究，候选人将有机会（i）给上层课程“哺乳动物生理学”的正式讲座，以及（ii）通过研究项目指导本科生。在候选人的拟议研究中，他将探索黄热病蚊子埃及伊蚊的Malpighian（肾）小管中的酸碱运输机制。他将确定这些小管是否代表了一个有效的模型系统，用于理解哺乳动物肾集合小管β-插入细胞中V型H+ ATP酶驱动的酸分泌。在拟议研究的第1年和第2年，候选人将使用分子方法克隆和定位马氏管中的酸碱转运蛋白，并将表征这些转运蛋白在异源表达系统（非洲爪蟾卵母细胞）以及天然小管上皮中的功能。在第3年，候选人将测量酸负荷对分离小管生理学的影响，并确定第1年和第2年确定的酸碱转运蛋白是否有助于跨上皮酸分泌。来自K 01奖的资金将允许候选人建立一个独立的项目，并被提名为康奈尔大学的研究助理教授职位。他也将有能力申请其他大学的独立职位。 相关性：V型H+ ATP酶（质子泵）是对人类健康至关重要的蛋白质。例如，质子泵基因缺陷的人通常会导致肾脏酸分泌受损、骨质疏松、耳聋和不育（男性）。该研究的目的是开发一个模型系统，以更好地了解质子泵在人类肾脏中的功能。这个模型系统是一种昆虫，黄热病蚊子的“肾脏”。尽管蚊子与人类有很大不同，但构成蚊子肾脏的细胞（i）与哺乳动物肾脏的酸分泌细胞非常相似，并且（ii）完全由质子泵提供能量。此外，蚊子肾脏比哺乳动物肾脏更容易在实验室中进行研究，使前者成为研究后者的理想实验模型系统。

DESCRIPTION (provided by applicant): Project Summary: This application proposes to transition the candidate (Peter Piermarini) from a postdoctoral scientist to an independent researcher. Fostering this transition are his mentors Professors Klaus Beyenbach and Michael Kotlikoff, and two external consultants (Boron, Yale and Harrison, Arizona State). In the proposed study, the candidate will (i) expand his experimental repertoire of molecular and genomic biology, (ii) acquire powerful methods in renal physiology, such as in vitro microperfusion of tubular epithelia, and (iii) gain a solid knowledge of electrophysiology. In addition to research, the candidate will have opportunities to (i) give formal lectures to the upper-level course 'Mammalian Physiology', and (ii) mentor an undergraduate student through a research project. In the candidate's proposed research, he will explore the mechanisms of acid-base transport in the Malpighian (renal) tubules of the yellow-fever mosquito, Aedes aegypti. He will determine if these tubules represent a valid model system for understanding V-type H+ATPase-driven acid secretion in (-intercalated cells of the mammalian, renal collecting tubule. In years 1 and 2 of the proposed study, the candidate will use molecular approaches to clone and localize acid-base transporters in Malpighian tubules, and will also characterize the function of these transporters in a heterologous expression system (Xenopus oocytes), as well as in the native tubule epithelium. In year 3, the candidate will measure the effects of an acid load on the physiology of isolated tubules, and determine if the acid-base transporters identified in years 1 and 2 contribute to transepithelial acid secretion. Funding from the K01 award will allow the candidate to establish an independent program and to be nominated for a research-track assistant professor position at Cornell. He also will be well positioned to apply for independent positions at other universities. Relevance: The V-type H+ATPase (proton pump) is a protein of vital importance to human health. For example, humans with genetic defects in the proton pump often have impaired acid secretion by the kidney, weakened bones, deafness, and sterility (in males). The goal of the proposed study is to develop a model system for better understanding the function of the proton pump in the human kidney. This model system is the 'kidney' of an insect, the yellow-fever mosquito. Although mosquitoes are quite different than humans, the cells that compose the kidneys of mosquitoes (i) closely resemble acid-secreting cells of the mammalian kidney, and (ii) are energized entirely by the proton pump. Moreover, mosquito kidneys are much more accessible and easier to study in the laboratory than mammalian kidneys, making the former an ideal experimental model system for studying the latter.