Saliva is the principal protective agent for the mouth and thus is of primary importance to oral health maintenance. Perturbations of salivary secretory mechanisms can consequently lead to serious oral health problems. The objective of this project is to study the membrane and cellular processes that underlie the phenomenon of salivary fluid secretion and thus to contribute to our understanding of the fluid secretory process. Because similar secretory mechanisms are thought to be common to a number of other tissues, this information should be of rather broad applicability and interest. During the present reporting period we have continued our in-depth studies of the salivary Na-K-2Cl cotransporter (NKCC1) and its homologues and in addition have begun to examine another family of transport proteins (the SLC26 anion transporters) that have recently been shown to play a significant role in exocrine salt and water secretion. ? ? 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. ? ? 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 this dimerization. Over the present reporting period we have continued to refine our information concerning the amino acids involved in the dimerization interaction. These experiments are being carried out by replacing regions and individual amino acids in the NKCC1 C-terminus with the corresponding amino acids from its close homologue NKCC2, with which NKCC1 does not dimerize. By testing for the dimerization of these chimeric and mutant proteins with wild-type NKCC1 we have been able to localize an essential dimerization motif consisting of 2-3 amino acids. Our results also suggest that neighboring regions, although not essential for dimerization, may play a role in generating a homologue-specific context for the interaction site. The effects of mutations associated with human disease that are found near the dimerization motif are also being investigated. ? ? In order to study the dimerization properties of our NKCC1/NKCC2 chimeras we have developed a specialized co-immunoprecipitation procedure that allows us to gently and specifically elute only one NKCC1 unit from immunoprecipitated NKCC1 dimer pairs. This eluate necessarily includes any other proteins associated with the eluted NKCC1 molecules. We are now using this procedure to screen for proteins that interact with NKCC1, beginning with several candidate proteins previously identified in our laboratory using yeast-two-hybrid screens.? ? The C-terminal 16 amino acids of NKCC1 contain a characteristic sequence that is highly conserved across species in many members of this gene family:? ILLVRGxxxxVLTxxx? where x denotes less well-conserved residues. Several observations led us to hypothesize that this sequence was involved in protein localization and/or expression. Accordingly we have systematically mutated these conserved C-terminal amino acids to investigate their potential role(s) in the behavior of NKCC1. Our results indicate that mutation of any two of the residues ILLV to alanine reduces both the expression and the complex glycosylation of NKCC1 and that multiple mutations to alanine have cumulative effects. Mutation of ILLV to AAAA reduces both expression and complex glycosylation by ~90%. By contrast, mutation or scrambling of other conserved C-terminal residues has little effect on NKCC1 expression or glycosylation. These results suggest that the sequence ILLV is required for export of NKCC1 from the endoplasmic reticulum to the Golgi apparatus. ? ? The so-called SLC26 family of anion transporters contains 11 mammalian members plus a number of homologues in lower species, including yeast and bacteria. Several members of this transporter family have been shown to play important roles in exocrine secretion in mammals. In addition, mutations in SLC26 transporters have been associated with a number of human genetic diseases including chondrodysplasias, chloride-losing diarrhea and hereditary deafness. Virtually nothing is known about the structure of this important family of membrane proteins. Over the past FY we have successfully functionally expressed a bacterial SLC26 homologue in E. coli. The ability to express this protein in E. coli provides us with a potentially powerful system for over-expression and purification of this protein for structural studies.
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