HCO3-transporters in Drosophila and Mosquitoes

细胞内和细胞外pH（酸碱转运）以及其他离子浓度（如Na+）的调节维持跨膜的离子梯度。正常的细胞功能是这些离子的向内和向外运动之间的平衡，这些离子通常响应于细胞内pH而变化。这在中枢神经系统、消化道、呼吸道和泌尿系统中尤其如此。几年前，我们使用表达克隆来克隆和表征肾生电Na/HCO3共转运蛋白（NBC）。在过去的4年中，我们和其他人已经发现了7 - 8组新的HCO3-转运蛋白在植物，脊椎动物和无脊椎动物。最近，我们克隆了一个Na+驱动的阴离子交换剂（NDAE 1）从果蝇和免疫定位NDAE 1上皮细胞（肠道，马氏管和唾液腺）以及中枢和外周神经系统。我们还鉴定并克隆了第二个果蝇HC03-转运蛋白，CG8177（塞雷拉符号）。我们推测HCO3-转运蛋白（NDAE 1和CG 8177）在双翅目昆虫上皮细胞和神经细胞的酸碱平衡和离子平衡中起重要作用。为了解决假设，我们提出了两个主要目标：首先，我们将使用果蝇遗传技术和我们的电生理学专业知识，以确定生理表型的NDAE 1突变体在生物体水平和特定的解剖组织。我们将使用果蝇中可用的遗传技术在内源性ndae 1基因中进行突变。我们的NDAE1抗体也适用于伊蚊。存在于伊蚊中的NDAE 1是令人兴奋的，因为生物体更大，使它们在生理上更容易实验。 不幸的是，目前在蚊子中可能进行的遗传和分子操作远少于果蝇。然而，在可能的情况下，我们将在伊蚊和按蚊及其组织中进行平行实验。其次，我们将阐明果蝇的另一种HCO3转运蛋白CG8177的定位和功能。我们的方法将类似于用于NDAE 1的方法，我们将在非洲爪蟾卵母细胞中表达编码CG8177的cRNA以确定转运的离子和离子亲和力。我们还将产生抗体以确定CG8177在果蝇中的组织和亚细胞定位。有趣的是，果蝇NDAE 1和哺乳动物NBC定位于功能类似的许多组织（肠、马氏管/肾、眼、脑），人类NBC和果蝇NDAE与人类突变引起的疾病有关。在蚊子中，碱性肠道Ph在感染性病原体的入侵中起作用，例如引起疟疾和黄热病的疟原虫。我们的方法相结合，适用于研究HCO3-转运蛋白在生物体中，可以遗传操纵将使我们能够确定这些HCO3-转运蛋白在几个重要组织中发挥的作用。因此，我们的建议将增加我们的理解双翅目酸碱平衡和耦合离子在几个组织，以及使未来的机会，以测试功能的相互作用假说在整个生物体。

Regulation of intracellular and extracellular Ph (acid-base transport), as well as other ionic concentrations, such as Na+, maintains ion gradients across membranes. Normal cell function is a balance between inward and outward movement of these ions often varying in response to intracellular pH. This is especially true in the central nervous system, digestive tract, respiratory tract, and urinary system. Several years ago, we used expression cloning to clone and characterize the renal electrogenic Na/HCO3 co-transporter (NBC). In the past 4 years, we and others have uncovered 7-8 groups of new HCO3-transporters in plants, vertebrates and invertebrates. Recently we have cloned a Na+-driven anion exchangers (NDAE1) from Drosophila and immunolocalized NDAE1 to epithelia (gut, Malpighian tubules and salivary glands) as well as the central and peripheral nervous system. We have also identified and cloned a second Drosophila HC03-transporter, CG8177 (Celera notation). We hypothesize that HCO3-transporters (NDAE1 and CG8177) play an important role in epithelial and neuronal acid-base and ionic homeostasis of Diptera. To address hypothesis, we propose to two major aims: First, we will use Drosophila genetic techniques and our electrophysiology expertise to determine the physiological phenotypes of NDAE1 mutants at the organismal level and in specific dissected tissues. We will use genetic techniques available in Drosophila to make mutations in the endogenous ndae1 gene. Our NDAE1 antibody also works in Aedes. NDAE1 being present in Aedes is exciting because the organisms are larger making them physiologically a more tractable experimentally. Unfortunately the genetic and molecular manipulations possible in mosquitoes are currently much less than those of Drosophila. However, where possible we will perform parallel experiments in Aedes and Anopheles and their tissues. Second, we will elucidate the localization and function of Drosophila other HCO3 transporter, CG8177. Our approach will be similar to that used for NDAE1, we will express the cRNA encoding CG8177 in Xenopus oocytes to determine the transported ions and ion affinities. We will also generate antibodies to determine the tissue and sub-cellular localization of CG8177 in Drosophila. Interestingly, Drosophila NDAE1 and mammalian NBC's localize to many tissues that are functionally analogous (gut, Malpighian tubules/kidney, eye, brain), human NBC and Drosophila NDAE are implicated in disease by human mutations. In mosquitoes an alkaline gut Ph plays a role in invasion by infectious agents, e.g. Plasmodium, which causes malaria and yellow fever. Our combination of approaches, applied to study HCO3-transporters in an organism which can be genetically manipulated will allow us to determine the roles these HCO3-transporters plays in several important tissues. Thus, our proposal will increase our understanding of Dipteran acid-base homeostasis and coupling ions in several tissues as well as enable future opportunities to test functional interaction hypotheses in whole organisms.