7. Project Summary Cation-chloride cotransporters (CCCs) regulate cell volume and intracellular chloride concentration in many different cell types. They contribute to salt secretion by epithelial cells in the lung, intestine, and sweat glands and salt absorption by epithelial cells in the kidney. They influence excitability of neurons in response to neurotransmitters such as gamma-aminobutyric acid and glycine by regulating the electrochemical gradient for chloride movement across the cell membrane. In mammals, the sodium-dependent CCCs include a potassium- independent sodium-chloride cotransporter (NCC) and two sodium-potassium-chloride cotransporters (NKCC1 and NKCC2). NCC and NKCC2 are targets of the clinically important thiazide and loop diuretics, whereas NKCC1 is a promising target for anti-epileptic drugs. Despite their physiological and medical importance, our understanding of the structure-function relationships of these transporters is incomplete. In this R15-AREA project, an undergraduate research group will evaluate the structure-function relationships of the NaCCC2s, a group of sodium-dependent CCCs that is specific to insects. The work will focus on Ncc83, a NaCCC2 from the fruit fly Drosophila melanogaster. According to preliminary data and published work, Ncc83 and its ortholog from the mosquito Aedes aegypti induce a chloride-independent sodium current when expressed in Xenopus oocytes. This activity contrasts with the electroneutral, chloride-dependent cation transport of other sodium- dependent cation-chloride cotransporters, including Drosophila Ncc69. The first aim characterizes the fundamental transport properties of Ncc83 such as ion and inhibitor affinities, testing the hypothesis that Ncc83 is a sodium transporter rather than a sodium channel. Ncc83 will be expressed in Xenopus oocytes and the insect Sf9 cell line and its activity assessed by two-electrode voltage clamp and tracer flux assays. Cation chromatography will be used to evaluate tracer levels of non-radioactive lithium (a sodium tracer) and rubidium (a potassium tracer). The second aim explores structure-function relationships through domain swap and site- directed mutagenesis experiments, testing the hypothesis that amino acid substitutions between Ncc83 and Ncc69 determine electrogenic versus electroneutral transport activity. Finally, the third aim tests the hypothesis that Ncc83 contributes to salt secretion and/or absorption by renal (Malpighian) tubules and hindgut. Using the genetic tools available for Drosophila, tissue-specific knockdowns of Ncc83 will be produced. Physiological effects will be assessed by sampling hemolymph following exposure to conditions that challenge ion homeostasis and by fluid secretion assays of tubules. Cation chromatography will be used to evaluate ion concentrations in fluids collected during these experiments. This project will uncover new information about the structure-function of CCCs, a medically important family of tr...