PHYSIOLOGY OF INSECT POTASSIUM ION TRANSPORT

长期目标是分析结构、功能和调节 的质膜H ~+ V-ATPase，分离出K ~+/2 H ~+ 并了解ATP酶-反向转运蛋白对的活性 使幼虫中肠腔碱化。 一个共同的目标是提供 利用cDNA探针和抗体控制媒介蚊虫的新基础 从运输模型，M。sexta研究中肠碱化， 疾病媒介模型，埃及伊蚊。 假设是高pH值的 毛虫和蚊子中肠都是由H+易位产生的， 质膜V-ATPase 质子动力的电压分量 激活顶端膜，并通过以下方式驱动电泳K+/H+交换： 一种新的K+/2 H+反向转运蛋白。 ATP酶的结构/功能分析 反向转运蛋白对本身是有价值的，并将提供分子 详细介绍了碱化机理。 选择性抑制 反向转运蛋白应防止蚊子中肠碱化， 杀死幼虫，对环境影响很小。 目的1是分析 V1复合物的结构和功能; ATP结合和水解， 以及亚基组装将使用重组亚基进行研究 通过体外转录/翻译或过表达产生; 最终，催化位点将被位点系统地修饰， 定向诱变 目的2分析亚基基因的调控 使用现有的基因组DNA文库表达;启动子元件将被 分离并在转染的细胞中监测其活性， 报告基因 目的3：利用蛋白质分离K+/2 H+逆向转运蛋白 生物化学以及克隆和测序其编码cDNA， 用脊椎动物Na+/H+逆向转运蛋白cDNA探针杂交克隆， 通过在Na+/H+反转运缺陷酵母中的互补克隆或通过 在非洲爪蟾卵母细胞中表达克隆。 目的4是分析 通过ATP酶-反向转运蛋白结构/功能比较研究碱化机理 在结构复杂的毛虫中肠中， 结构简单的蚊子中肠， 免疫细胞化学和评价碱化封闭与 微胶囊化的阿米洛利衍生物。 该提案具有科学价值 因为质子动力会破坏动物细胞质膜， 二次活性剂的酸化作用及酸化方向逆转 阳离子/质子反向转运都是新颖的概念。 与健康相关 因为新的基础科学可以立即应用于疾病媒介 模型

The long term objective is to analyse the structure, function regulation of the plasma membrane H+ V-ATPase in Manduca sexta, to isolate the k+/2H+ antiporter and to understand how activity of the ATPase-antiporter couple alkalinizes the larval midgut lumen. A concomitant objective is to provide a new basis for vector mosquito control by using cDNA probes and antibodies from the transport model, M. sexta to study midgut alkalinization in a disease vector model, Aedes aegypti. The hypothesis is that the high pH of both caterpillar and mosquito midgut is generated by an H+ translocating, plasma membrane V-ATPase. The voltage component of the protonmotive force energized the apical membrane and drives electrophoretic K+/H+ exchange by a novel K+/2H+ antiporter. A structure/function analysis of the ATPase- antiporter couple will be worthwhile in itself and will provide molecular details of the alkalinization mechanism. Selective inhibition of the antiporter should prevent mosquito midgut alkalinization, eventually killing larvae with little environmental impact. Aim 1 is to analyse the structure and function of the V1 complex; ATP binding and hydrolysis as well as subunit assembly will be studied using recombinant subunits produced by in vitro transcription/translation or over-expression; eventually the catalytic site will be systematically modified by site directed mutagenesis. Aim 2 is to analyse the regulation of subunit gene expression using an existing genomic DNA library; promoter elements will be isolated and their activity will be monitored in transfected cells using reporter genes. Aim 3 is to isolate the K+/2H+ antiporter by protein biochemistry as well as to clone and sequence its encoding cDNA by hybridization cloning with cDNA probes from vertebrate Na+/H+ antiporter, by complementation cloning in Na+/H+ antiport-deficient yeast or by expression cloning in Xenopus oocytes. Aim 4 is to analyze the alkalinization mechanism by comparing ATPase-antiporter structure/function in the structurally complex caterpillar midgut with that in the structurally simple mosquito midgut using in situ hybridization and immunocytochemistry and to evaluate alkalinization blocking with microencapsulated amiloride derivatives. The proposal has SCIENTIFIC MERIT because energization of animal cell plasma membranes by a protonmotive force and reversal of acidification direction by secondary active cation/proton antiport are both novel concepts. It has HEALTH RELEVANCE because the new basic science is immediately applied to a disease vector model.