Malpighian Tubule Transport Physiology: Mechanism and Regulation

这项提议旨在利用遗传学、分子生物学和经典生理学来研究昆虫肾脏的马氏管上皮运输问题。首席研究员Beyenbach在实验室对成年蚊子的马氏管进行的研究发现了新的运输现象，这些现象刺激了对昆虫以外生物系统中上皮运输的研究。例如，他在马氏小管中的发现a)V型H+ATPase而不是Na/K ATPase为不同的上皮运输系统提供能量，b)分隔(紧密)连接的通道样行为，以及c)在惊人的时间域中对细胞旁通透性的快速调节，这些都促使人们在脊椎动物组织中寻找类似的现象。本提案是上述调查结果的合乎逻辑的延伸。假说驱动的实验有望发现新的运输系统，同时探索运输生理学的新想法。在目标1中，他已经在马氏伊蚊小管中克隆了NHE8(一种Na-H转运蛋白)的功能特征，这可能揭示出任何组织中第一个电生的Na/H交换器，无论是脊椎动物还是无脊椎动物。目的2应用蛋白质组学和高灵敏的质谱学方法，不仅可以鉴定NHE8，还可以鉴定新的蛋白质，以及可能在成人马氏管顶膜上重要的新转运蛋白。在目标3中，他试图a)识别缝隙连接，b)确定缝隙连接的大小和电荷选择性，c)探索缝隙连接在细胞间钙波传递中的作用，这可能介导亮氨酸激动素的信号转导，以及d)测试缝隙连接代谢调节的新想法。在分子生物学(Wieczorek)、细胞内钙成像(Walz)和蛋白质组学(Schooley)领域拥有国际声誉的三位合作者已经承诺他们的实验室将致力于该项目的成功。推动这一合作的是从不同角度阐明重要研究问题所产生的深刻理解。更广泛的影响。PI是一位热情的老师和导师，他的实验室吸引了一些最有天赋和最有动力的本科生。许多人毕生致力于研究。在他的实验室里，多达46名本科生与研究生和博士后一起接受了培训；19名本科生在参考期刊上共同撰写了论文。许多本科生已经被医学博士项目录取，一些人现在已经拥有了自己的研究实验室。想知道是一种强大的动力，也许是PI和他的学生最强烈的动力。PI在校园里为高年级学生和研究生教授一门很受欢迎的选修课--哺乳动物生理学。他是康奈尔大学本科生生理学研究项目的主任。PI积极参与国际教育，帮助美国学生在德国获得暑期研究职位，并帮助德国学生在美国找到研究机会或临床轮换。PI是美国亚历山大·冯·洪堡协会(www.avhaa.org)的创始成员之一。由于促进了教育和研究领域的国际合作，他获得了德国最高文职人员奖--德国联邦教育奖。

This proposal aims to bring genetics, molecular biology and classical physiology to bear on questions of epithelial transport in Malpighian tubules, the kidneys of insects. Studies of adult mosquito Malpighian tubules in the lab of the principal investigator Beyenbach have uncovered new transport phenomena that stimulate research of epithelial transport in biological systems beyond insects. For example, his finding in Malpighian tubules of a) the energizing of diverse epithelial transport systems with the V-type H+ ATPase and not the Na/K ATPase, b) the channel-like behavior of septate (tight) junctions, and c) the rapid regulation of paracellular permeability in the astonishing time domain of seconds, are motivating searches for similar phenomena in vertebrate tissues. The present proposal is a logical extension of the above findings. Hypothesis-driven experiments promise to discover new transport systems while exploring new ideas in transport physiology. In Aim 1, the functional characterization of NHE8 (a Na-H transport protein) he has already cloned in Aedes Malpighian tubules may uncover the first electrogenic Na/H exchanger in any tissue, vertebrate or invertebrate. Aim 2 applies proteomics and highly sensitive mass spec methods with the promise of identifying not only NHE8 but also new proteins, and potentially important new transporters in the apical membrane of the adult Malpighian tubule. In Aim 3 he seeks to a) identify gap junctions, b) determine size and charge selectivity, c) explore the role of gap junctions in cell-to-cell transmission of Ca2+ waves that may mediate the signal transduction of leucokinin, and d) test the novel idea of the metabolic regulation of gap junctions. Three collaborators with an international reputation in molecular biology (Wieczorek), intracellular Ca2+ imaging (Walz), and proteomics (Schooley) have committed their labs to the success of this project. What has motivated this collaboration is the deep understanding that derives from illuminating important research questions from diverse perspectives. Broader Impact. The PI is an enthusiastic teacher and mentor whose lab attracts some of the most gifted and motivated undergraduates. Many make a lifetime commitment to research. As many as 46 undergraduates have been trained in his lab alongside graduate students and post-docs; 19 undergraduates share authorship in refereed journals. Many undergraduates have been accepted in MD-PhD programs, and some have now their own research laboratories. Wanting to know is a powerful drive, perhaps the strongest motivation of the PI and his students. The PI teaches a popular elective Biology course on campus- Mammalian Physiology -to seniors and graduate students. He serves as Director of the Cornell program of study Undergraduate Physiology. The PI is active in international education, helping US students secure summer research positions in Germany, and assisting German students find research opportunities or clinical rotations in the US. The PI is a founding member of the Alexander von Humboldt Association of America (www.avhaa.org). He has received the highest German civilian award, the Bundesverdienstkreuz, for fostering international cooperation in education and research.