During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1) and its homologues. NKCC1 belongs to a small gene family (SLC12) with nine homologues in vertebrates. Of these, seven are known to be electroneutral cation-chloride cotransporters while the function of the remaining two vertebrate homologues remains uncertain. NKCC1 is relatively widely expressed in both epithelial and non-epithelial tissues and is known to play important roles in a variety of physiological processes including transepithelial salt and water transport, hearing, olfaction, pain perception, spermatogenesis, and maintenance of blood pressure and vascular tone. NKCC1 is the major Cl entry pathway into salivary acinar cells and thus is primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of the structure/function relationships of this protein and its behavior in acinar cells will greatly improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. In past studies we established that the functional unit of NKCC1 is a homodimer and that the intracellular 450 amino acid C-terminus of the protein is largely responsible for its dimerization. We have been employing chemical crosslinking studies and a novel co-immunoprecipitation assay to identify and characterize the amino acids involved in the dimerization interaction. Our results indicate that several regions within the C-terminus play a role in dimerization. Replacement of these amino acids with the corresponding residues from NKCC2 (a close homologue which also forms homodimers but does not dimerize with NKCC1) results in chimeras or point mutants that have weakened or undetectable dimerization interactions with wild-type NKCC1. In particular we have found a single glycine reside whose replacement with cysteine (the corresponding amino acid in NKCC2), serine or threonine results in mutants that do not dimerize with wild-type NKCC1. These point mutants also have weakened homo-dimerization reactions with themselves. We conclude that this glycine and the surrounding amino acids play a central role in the conformation of the dimer interface and we have suggested a simple schematic model for the dimerization interaction that can account for our observations. In Drosophila there are 5 NKCC1 homologues including at least one member on each of the 4 main branches of the vertebrate SLC12 phylogenetic tree. In early 2007 we began a project aimed at characterizing the Drosophila SLC12 proteins (in collaboration with K. Ten Hagen). Our goal here was to attempt to understand the developmental and functional roles of these proteins in Drosophila and to ultimately use this information to better understand the properties of their vertebrate homologues. We have employed in situ hybridization to study the expression patterns of the Drosophila SLC12 proteins during embryonic development. Our studies indicate that all five members of this family are expressed during early embryogenesis (stages 1-6) but that spatial and temporal expression patterns become more refined as development proceeds. Expression during late embryogenesis was seen predominantly in the ventral nerve cord, salivary gland, gut and anal pad. In parallel studies we have carried out transport assays on each of the five Drosophila homologues expressed as recombinant proteins in the cultured insect cell line High Five. Under our experimental conditions we found that only one of these proteins, CG4357, transported the potassium congener 86Rb. Additional experiments established that 86Rb transport via CG4357 was saturable, sodium-dependent, chloride-dependent and potently inhibited by bumetanide, a specific inhibitor of vertebrate Na-K-2Cl cotransporters. The kinetic properties of CG4357 are remarkably similar to those found previously in our laboratory for NKCC1. Thus these results indicate that CG4357 is an insect orthologue of the vertebrate Na-K-2Cl cotransporters. Given the ease-of-use and productivity of insect expression systems we anticipate that CG4357 will be a strong candidate for future structural studies of the SLC12 proteins.