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. NKCC1 is also an important candidate gene for the treatment of salivary hypofunction via gene transfer in our Branch and we are collaborating with Dr. Bruce Baum on some initial experiments related to this. ? ? In past studies we have 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 dimerization interactions with wild-type NKCC1. In particular we have found a single glycine residue whose replacement with other small amino acids such as serine, cysteine or alanine results in mutants that form homodimers but do not dimerize with wild-type NKCC1. These complementary effects of relatively conservative mutations of a single amino acid were quite unexpected. Our conclusion is that this glycine must play an important role in the conformation of the dimer interface. The final experiments required to complete this project are now being carried out.? ? 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 these proteins during Drosophila embryogenesis. Our studies indicate that all five members of this family are expressed early in embryogenesis (stages 1-6) but spatial and temporal expression patterns become more refined as development proceeds. Expression became more restricted during mid-embryogenesis (stages 7-12), as only three family members were expressed in the developing gut. Expression during late embryogenesis was seen predominantly in the ventral nerve, brain, salivary gland, and posterior spiracles. We have raised antibodies against these proteins and additional experiments designed to characterize SLC12 protein expression in the Drosophila adult are planned. In parallel studies we are carrying out transport assays on each of the five Drosophila homologues expressed as recombinant proteins in the cultured insect cell line High Five.
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