Collaborative Research: Na,K,Cl Cotransporters in Mosquito Epithelial Transport - Connecting Molecules to Physiology

合作研究：蚊子上皮转运中的 Na、K、Cl 协同转运蛋白 - 连接分子与生理学

• 批准号: 1557298
• 负责人: Peter Piermarini
• 金额: $ 8.1万
• 依托单位: Ohio State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1557298&HistoricalAwards=false
• 关键词: Collaborative; Research; Na; Cl; Cotransporters

This research explores the mechanisms that mosquitoes use to regulate their salt and water balance under the challenging conditions they face during their lifecycles. Most mosquito larvae live in freshwater and must absorb salt to counteract the tendency for salt to diffuse out of their bodies. In contrast, adult female mosquitoes must expel large amounts of salt after they engorge on a salty blood meal. The specific focus of this project is on three mosquito proteins that help carry salts such as sodium, potassium, and chloride into or out of the body. One of these proteins is closely related to proteins in humans and other vertebrate animals that are important to salt and water balance. Thus, studying the mosquito version of this protein may uncover fundamental properties of salt-transporting proteins that are shared between vertebrates and insects. The other two proteins are only found in mosquitoes and other insects, and have not been characterized in any animal. Gaining a better understanding of these novel insect proteins may be especially useful in developing strategies to control mosquitoes and other insect pests, since it may be possible to target them without affecting vertebrate proteins. This research project will develop new tools for researchers to assess where these proteins are located in the mosquito body, what salts they carry, and what chemicals can interfere with their function. Additionally, three undergraduate students and one graduate student will receive closely mentored research experiences. Mosquitoes must secrete and absorb ions differently depending on their life stage, sex, and environment. Three proteins from the yellow fever mosquito Aedes aegypti have sequence similarity to vertebrate Na+-dependent cation-chloride cotransporters (CCCs), which participate in both ion absorption and secretion. This work will produce critical reagents and refine procedures that are necessary to begin linking the molecular properties of the mosquito CCCs to their transport functions and whole-animal physiological roles. The first objective is to develop isoform-specific antibodies that recognize and differentiate among each of the three mosquito CCCs. The selectivity of the antibodies will be evaluated by comparing their reactivity among different mosquito tissues and developmental stages, mosquitoes in which expression of a specific CCC has been silenced by RNA interference, and in Xenopus oocytes injected with cRNAs encoding a particular CCC. The second objective is to confirm functional heterologous expression of each mosquito CCC in Xenopus oocytes. Functional expression of the CCCs will be evaluated using standard radioisotope uptake assays and newly developed non-radioactive methods. Findings from the proposed work may also be useful in identifying novel molecular targets to aid in the development of new insecticides for the control of insect disease vectors and agricultural pests. Three undergraduate students and one graduate student will receive mentored research training and the principal investigators will initiate community outreach programs.

这项研究探索了蚊子在其生命周期中面临的挑战性条件下用于调节其盐和水平衡的机制。 大多数蚊子幼虫生活在淡水中，必须吸收盐来抵消盐从体内扩散出去的趋势。 相比之下，成年雌蚊在饱食含盐的血餐后必须排出大量的盐。 该项目的具体重点是三种蚊子蛋白质，这些蛋白质有助于将钠，钾和氯等盐带入或带出身体。 其中一种蛋白质与人类和其他脊椎动物中对盐和水平衡很重要的蛋白质密切相关。因此，研究这种蛋白质的蚊子版本可能会揭示脊椎动物和昆虫之间共享的盐转运蛋白的基本特性。另外两种蛋白质只在蚊子和其他昆虫中发现，尚未在任何动物中发现。更好地了解这些新的昆虫蛋白质可能对开发控制蚊子和其他昆虫害虫的策略特别有用，因为有可能在不影响脊椎动物蛋白质的情况下靶向它们。该研究项目将为研究人员开发新的工具，以评估这些蛋白质在蚊子体内的位置，它们携带的盐以及哪些化学物质会干扰它们的功能。此外，三名本科生和一名研究生将获得密切指导的研究经验。 蚊子必须根据其生命阶段、性别和环境而分泌和吸收不同的离子。来自黄热病蚊子埃及伊蚊的三种蛋白质与脊椎动物Na+依赖性阳离子-氯化物协同转运蛋白（CCCs）具有序列相似性，CCCs参与离子吸收和分泌。这项工作将产生关键试剂和完善程序，这是开始将蚊子CCC的分子特性与其运输功能和整个动物的生理作用联系起来所必需的。第一个目标是开发同种型特异性抗体，识别和区分三种蚊子CCC中的每一种。通过比较不同蚊子组织和发育阶段、特定CCC的表达已通过RNA干扰沉默的蚊子和注射编码特定CCC的cRNA的爪蟾卵母细胞中的抗体反应性来评价抗体的选择性。第二个目的是确认每个蚊子CCC在爪蟾卵母细胞中的功能性异源表达。将使用标准放射性同位素摄取测定和新开发的非放射性方法评价CCC的功能表达。这项工作的发现也可能有助于确定新的分子靶点，以帮助开发新的杀虫剂来控制昆虫疾病媒介和农业害虫。三名本科生和一名研究生将接受指导研究培训，主要研究人员将启动社区外展计划。