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

描述(由申请人提供)： 项目摘要：该应用程序建议将候选人(Peter Piermarini)从博士后科学家转变为独立研究人员。推动这一转变的是他的导师克劳斯·贝延巴赫教授和迈克尔·科特利科夫教授，以及两名外部顾问(亚利桑那州立大学的博龙和哈里森)。在拟议的研究中，候选人将(I)扩大他的分子和基因组生物学实验体系，(Ii)获得肾脏生理学的强大方法，如体外肾小管上皮微灌流，以及(Iii)获得扎实的电生理学知识。除了研究，候选人还将有机会(I)给高级课程《哺乳动物生理学》做正式讲座，以及(Ii)通过研究项目指导本科生。在候选人提出的研究中，他将探索黄热病蚊子埃及伊蚊的马氏管(肾脏)酸碱运输的机制。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V型H+ATPase驱动的哺乳动物肾脏集合管嵌入细胞中的酸分泌。在拟议研究的第一年和第二年，候选人将使用分子方法克隆和定位马氏小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(非洲爪哇卵母细胞)以及天然小管上皮中的功能。在第三年，候选人将测量酸负荷对孤立小管生理的影响，并确定第一年和第二年确定的酸碱转运体是否有助于跨上皮酸的分泌。K01奖项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研究跟踪助理教授的职位。他还将处于有利地位，可以申请其他大学的独立职位。 相关性：V型H+ATPase(质子泵)是一种对人类健康至关重要的蛋白质。例如，具有质子泵基因缺陷的人类通常会损害肾脏的酸分泌，骨骼变弱，耳聋和不育(男性)。这项拟议研究的目标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”。尽管蚊子与人类有很大的不同，但组成蚊子肾脏的细胞(I)与哺乳动物肾脏的酸性分泌细胞非常相似，(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获得，也更容易在实验室进行研究，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.