PHYSIOLOGY OF INSECT POTASSIUM TRANSPORT

总体目标是表征鳞翅目昆虫中肠K（+）稳态; 根据中肠模型，K（+）通过中肠细胞中的通道进入中肠细胞。 基底膜，并被推动穿过杯状细胞顶膜，GCAM， 通过一个与一个平行的生电H（+）泵， nH（+）/K（+）反向转运蛋白;顶膜被通电至PD >180 mV。 与7.0的细胞pH相比，PD支持管腔pH >10;它驱动 氨基酸/K（+）从管腔向柱状细胞共输，导致氨基酸/K（+）从管腔向柱状细胞共输， 酸吸收和完成K（+）稳态循环。 此续订 应用程序处理GCAM。假设是质子泵 产电V-ATP酶和插入的产电nH（+）/K（+）反向转运蛋白 转化为阳离子不可渗透的脂质双层是 稳态途径 目的一是完成GCAM基因的全序列测定 67和56 kDa的V-ATP酶亚基，并克隆和测序43， 28和16 kDa亚基。 目的二是溶解和纯化反向转运蛋白 蛋白质。 目的3是克隆和测序编码 反向转运蛋白 目的4：定位V-ATP酶和nH（+）/K（+） K（+）转运上皮细胞的反向转运蛋白，并描述了GCAM的个体发生 从液泡膜中分离出来。 GCAM将通过超声处理和梯度分离 离心法 反向转运蛋白将被非离子去污剂溶解 并通过密度梯度离心和/或FPLC纯化， 重构为脂质体作为测定。 中肠幼虫cDNA文库 筛查将使用中肠蛋白抗体或寡核苷酸 从其他来源的高度同源组分的探针。 atp酶和 反向转运蛋白定位将使用荧光和金标记抗体 在光和EM免疫细胞化学中适当的亚基蛋白。 中肠模型可为研究血浆V-ATP酶活力提供新的思路 其他昆虫的膜和脊椎动物的尿道， 骨膜 因为中肠模型处理一种不寻常的阳离子 保护细胞不受高碱性内腔影响的不可渗透的顶端膜， 它提供了对苏云金芽孢杆菌作用的深入了解， 内毒素;它可能导致其他环境安全的毛虫， 蚊幼虫控制。

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.