PHYSIOLOGY OF INSECT POTASSIUM TRANSPORT

总体目标是描述鳞翅目昆虫中肠K()的动态平衡； 根据中肠模型，K()通过中肠中的通道进入中肠细胞。 并被推进穿过杯状细胞顶膜，GCAM， 通过一个电生H()泵与一个 NH()/K()逆向转运蛋白；顶膜通电至PD&GT；180 mV。 PD支持管腔的pH值为10，而细胞的pH值为7.0；它驱动 氨基酸/K()从管腔向柱状细胞运输，产生氨基酸 酸吸收和完成K()动态平衡循环。此次续订 应用程序处理GCAM。假设是一个质子泵浦 插入生电V-ATPase和生电NH()/K()逆向转运蛋白 进入阳离子不透性脂质双层的关键成分 动态平衡途径。目标1是完成编码GCAM的cDNA测序 67 kDa和56 kDa的V-ATPase亚基以及克隆和测序43的cDNA， 28和16 kDa亚基。目标2是溶解和纯化反向转运蛋白。 蛋白质(S)。目的3是克隆和测序编码该基因的基因 反向转运者(S)。目的4是定位V-ATPase和NH()/K() K()转运上皮中的逆向转运蛋白及其对GCAM个体发育的描述 来自液泡膜。GCAM将通过超声波和梯度分离 离心法。反转运蛋白将被非离子洗涤剂增溶 并通过密度梯度离心法和/或FPLC使用 重组成脂质体作为一种检测。中肠幼虫cDNA文库 筛查将使用中肠蛋白抗体或寡核苷酸 探查来自其他来源的高度同源成分。ATPase和 反向转运蛋白定位将使用荧光和金标抗体 在光和EM免疫细胞化学中选择合适的亚单位蛋白质。 中肠模型可能提供对血浆V-ATPase能量化的洞察 其他昆虫的膜和脊椎动物尿液和尿液的能量 骨膜。因为中肠模型处理了一种不寻常的阳离子 不透性的根尖膜保护细胞免受高度碱性管腔的伤害， 它为苏云金芽孢杆菌的作用提供了洞察力 内毒素；它可能会导致其他环境安全的毛虫和 蚊子幼虫控制。

The overall goal is to characterize lepidopteran midgut K(+) homeostasis; according to the midgut model, K(+) enters midgut cells via channels in the basal membranes and is propelled across goblet cell apical membrane, GCAM, to goblet cavity via an electrogenic H(+) pump in parallel with an nH(+)/K(+) antiporter; the apical membrane is energized to a PD >180 mV. The PD supports a lumen pH >10 compared to the cell pH of 7.0; it drives amino acid/K(+) symport from lumen to columnar cells, resulting in amino acid absorption and completing the K(+) homeostatic cycle. This renewal application deals with GCAM. The hypothesis is that a proton pumping electrogenic V-ATPase and an electrogenic nH(+)/K(+) antiporter inserted into a cation impermeable lipid bilayer are key components of the homeostatic pathway. Aim 1 is to complete sequencing cDNAs encoding GCAM V-ATPase subunits of 67 and 56 kDa and to clone and sequence cDNAs for 43, 28, and 16 kDa subunits. Aim 2 is to solubilize and purify the antiporter protein(s). Aim 3 is to clone and sequence the cDNA encoding the antiporter(s). Aim 4 is to localize the V-ATPase and nH(+) /K(+) antiporter in K(+) transporting epithelia and describe the ontogeny of GCAM from vacuolar membranes. GCAM will be isolated by sonication and gradient centrifugation. Antiporter will be solubilized with nonionic detergents and purified by density gradient centrifugation and/or FPLC using reconstitution into liposomes as an assay. Midgut larval cDNA library screening will use either antibodies to midgut proteins or oligonucleotide probes to highly homologous components from other sources. ATPase and antiporter localization will use fluorescent and gold labelled antibodies to appropriate subunit proteins in both light and EM immunocytochemistry. The midgut model may provide insight into V-ATPase energization of plasma membranes of other insects and into energization of vertebrate urinary and bone membranes. Because the midgut model deals with an unusual cation impermeable apical membrane protecting cells from a highly alkaline lumen, it has provided insight into the action of the Bacillus thuringiensis endotoxin; it may lead to other environmentally safe caterpillar and mosquito larval controls.