PHYSIOLOGY OF INSECT AMINO ACID TRANSPORT

昆虫氨基酸运输的生理学

• 批准号: 2065620
• 负责人: WILLIAM R HARVEY
• 金额: $ 21.04万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-12-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2065620
• 关键词: Aedes; Insecta; Manduca; Xenopus oocyte; aminoacid transport; brush border membrane; disease vectors; homeostasis; immunocytochemistry; in situ hybridization; insect control; ionophores; larva; membrane transport proteins; molecular cloning; physiology; protein structure function

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.

长期目标是比较主要结构和 氨基酸/碱金属离子分类蛋白的功能特性 甘达·塞克斯塔（Manduca Sexta）碱性中肠和埃及埃及埃及的蛋白质，到 分析了分析在化学上耦合氨基酸中的作用 吸收并利用这种不寻常的象征来发展新的， 环境安全的蚊子。 假设是高 毛毛虫和蚊子幼虫中肠的碱度不断 由新型的V-ATPase生成 通过施加跨膜质子电化学梯度来膜， delta/mu/h+，管腔阳性。 delta/mu/h+由k+/nh+转换 对K+电化学梯度的抗管剂，delta/mu/k+，提高了 富含碳酸盐的管腔pH至> 10.5。 delta/mu/k+，主要表示 Delta/psi，化学上的化学驱动氨基酸（AA）吸收碱的吸收 改编的AA/K+分类器（请参见下面的草图）。 AIM 1是通过快速表征甘氨酸/K+和脯氨酸/K+共线 刷子边界膜囊泡的过滤研究。 目标2是 序列M. sexta分类器cDNA克隆，通过注射筛选 从喂养幼虫到爪蟾的转录大小的mRNA分数 卵母细胞。 AIM 3是将M.分离和序列分类器cDNA克隆分离。 Sexta和Ae。与Xenic的杂交 编码脊椎动物肾脏，脑的已知氨基酸共菌的cDNA 和其他组织。 AIM 4是重组纯化，重组 对称性脂质体的伴侣，并以此为特征 离子特异性，动力学参数和底物选择性，使用 氨基酸的快速过滤和荧光猝灭测量值 吸收。 AIM 5是将氨基酸/K+分类器定位在中肠中 M. Sexta和Ae。埃及通过免疫细胞化学和原位 杂交。 科学优点：使用ATP水解，化学上的化学效果 delta/mu/h+和delta/mu/k+，要驱动氨基酸摄取是第一个 这种耦合在动物细胞质膜中的示例。 健康 相关性：如果幼虫AE。埃及中肠也被充满活力 通过跨膜膜三角/mu/h+进行化学运动转化为 delta/mu/k+，如果不寻常的aa/k+伴侣已经进化 由此产生的疾病载体dipteran midguts的碱性环境 哪些干扰不寻常的伴侣可以作为安全的发展 蚊子。