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.
|Effective start/end date||7/1/02 → 6/30/04|