PHYSIOLOGY OF INSECT POTASSIUM ION TRANSPORT

长期目标是分析其结构、功能和调节 六叶藻质膜H+V-ATPase的分离，以分离K+/2H+ 以及了解ATPase-反向转运蛋白对的活性 使幼虫的中肠腔碱化。随之而来的目标是提供 利用cDNA探针和抗体进行媒介灭蚊的新依据 从转运模型出发，六分枝杆菌研究了一种 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通过H+移位产生的， 质膜V-ATPase。质子动势的电压分量 使顶膜通电并驱动K+/H+电泳性交换 一种新的K+/2H+逆向转运蛋白。ATPase的结构/功能分析 反向转运子对本身将是有价值的，并将提供分子 碱化机理的详细信息。选择性抑制 逆向转运蛋白最终应该防止蚊子中肠的碱化 杀灭幼虫，对环境影响小。目标1是分析 V1复合体的结构和功能；ATP结合和水解AS 以及使用重组亚基组装亚基的研究 通过体外转录/翻译或过度表达产生； 最终，催化位将按位置进行系统的修饰 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文库进行表达；启动子元素将 它们的活性将在转基因细胞中进行监测 记者基因。目的3：通过蛋白质分离K+/2H+逆向转运蛋白 并对其编码基因进行克隆和测序。 脊椎动物Na+/H+逆向转运蛋白基因探针的杂交克隆， 通过在Na+/H+反转运缺陷酵母中进行互补克隆或通过 非洲爪哇卵母细胞的表达克隆。目标4是分析 比较ATPase-逆向转运蛋白结构/功能的碱化机制 在结构复杂的毛虫中肠中与在 结构简单的蚊子中肠的原位杂交和 免疫细胞化学和评价碱化阻滞剂 微胶囊阿米洛利衍生物。这项建议具有科学价值。 因为通过质子动力使动物细胞质膜充满能量 二次活动对酸化方向的作用力和反转作用 阳离子/质子反键都是新概念。它与健康相关 因为新的基础科学立即应用于疾病媒介 模特。

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.