PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

长期目标是比较一级结构和 氨基酸/碱金属离子同向转运体的功能特性 来自天蛾和埃及伊蚊的碱性中肠的蛋白质， 分析了共转运体在化学渗透偶联氨基酸中的作用， 吸收并利用这种不寻常的共生体来开发新的， 对环境安全的灭蚊剂。 假设是， 毛虫和蚊幼虫中肠的碱性持续 由一种新型的V-ATP酶产生，该酶利用ATP为顶端细胞提供能量， 膜通过施加跨膜质子电化学梯度， δ/mu/H+，管腔阳性。 δ/mu/H+转换为K+/nH+ K+电化学梯度的反向转运蛋白，delta/mu/K+，提高了 富含碳酸盐的内腔pH值>10.5。 δ/mu/K+，主要表示为 δ/psi，化学渗透驱动碱吸收氨基酸（AA） 适应AA/K+共转运蛋白（见下图）。 目的1：利用快速荧光定量PCR技术， 在刷状缘膜囊泡中的过滤研究。 目标2是 序列M. sexta同向转运体cDNA克隆，通过注射 转录的大小mRNA片段，从喂养的幼虫到爪蟾 卵母细胞 目的3：分离并测序M. Sexta和Ae.异源杂交构建埃及中肠cDNA文库 编码已知来自脊椎动物肾、脑、 和其他组织。 目的4是重组纯化的， 共转运体到功能性脂质体中，并根据 离子特异性、动力学参数和底物选择性， 氨基酸的快速过滤和荧光猝灭法 摄取。 目的5是定位氨基酸/K+共转运体在中肠， M. Sexta和Ae.免疫细胞化学和原位 杂交方法 科学优点：使用ATP水解，通过化学渗透偶联 δ/mu/H+和δ/mu/K+，驱动氨基酸摄取是第一位的 在动物细胞质膜中的这种偶联的实例。 健康 相关性：如果幼虫Ae.埃及中肠也充满活力 通过经心尖膜δ/mu/H+转化为 δ/mu/K+，如果不寻常的AA/K+共转运体已经在 产生的碱性环境的疾病媒介双翅目昆虫中肠然后剂 干扰不寻常的共转运蛋白的基因可以被开发为安全的 灭蚊剂

The LONG TERM OBJECTIVE is to compare the primary structure and functional characteristics of amino acid/alkali metal ion symporter proteins from alkaline midguts of Manduca sexta and Aedes aegypti, to analyses the symporters's roles in chemiosmotically coupled amino acid uptake and to exploit this unusual symport to develop new, environmentally safe mosquitocides. The HYPOTHESIS is that the high alkalinity of caterpillar and mosquito larval midgut is continuously generated by a novel V-ATPase that uses ATP to energize the apical cell membrane by imposing a transmembrane proton electrochemical gradient, delta/mu/H+, lumen positive. The delta/mu/H+ is transformed by a K+/nH+ antiporter to a K+ electrochemical gradient, delta/mu/K+, raising the carbonate-rich lumen pH to >10.5. delta/mu/K+, expressed mainly as delta/psi, chemiosmotically drives amino acid (AA) uptake by alkali adapted AA/K+ symporters (see sketch below). AIM 1 is to characterize glycine/K+ and proline/K+ symporters by rapid filtration studies in brush border membrane vesicles. AIM 2 is to sequence M. sexta symporter cDNA clones, screened by injection of transcribed, sized mRNA fractions from feeding larvae into Xenopus oocytes. AIM 3 is to isolate and sequence symporter cDNA clones from M. sexta and Ae. aegypti midgut cDNA libraries by hybridization with xenic cDNAs encoding known amino acid symporters from vertebrate kidney, brain and other tissues. AIM 4 is to reconstitute purified, recombinant symporters into functional liposomes and characterize them in terms of ionic specificities, kinetic parameters and substrate selectivity, using rapid filtration and fluorescence quenching measurements of amino acid uptake. AIM 5 is to localize amino acid/K+ symporters in midguts from M. sexta and Ae. aegypti by immunocytochemistry and in situ hybridization. SCIENTIFIC MERIT: The use of ATP hydrolysis, chemiosmotically coupled by delta/mu/H+ and delta/mu/K+, to drive amino acid uptake is the first example of such coupling in animal cell plasma membranes. HEALTH RELEVANCE: If larval Ae. aegypti midguts are also energized chemiosmotically by transapical membrane delta/mu/H+ transformed to delta/mu/K+, and if unusual AA/K+ symporters have evolved in the resulting alkaline milieu of disease vector dipteran midguts then agents which interfere with the unusual symporters can be developed as safe mosquitocides.