Saliva is the principle 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 secre-tion and thus to contribute to our under-standing 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 function, regulation and molecular biology of the salivary Na-K-2Cl cotransporter. This plasma membrane transport protein is thought to be the major Cl entry pathway into salivary acinar cells and thus to be primarily responsible for driving Cl secretion, and thereby fluid secretion, in salivary glands. Obtaining a better understanding of this protein and its behavior in acinar cells will improve our knowledge of salivary function and dysfunction, as well as possibly providing indications of how to treat the latter. Over the past year we have concentrated on three projects: (i) Identifying the phosphorylation site(s) associated with upregulation of cotransporter activity in response to $-adrenergic stimulation. Our previous data suggests that this phosphorylation is due to protein kinase A and that only the phosphoprotein is functionally active. During the present reporting period we have localized this phosphorylation site to an N-terminal peptide obtained by CnBr digestion. Interestingly, this peptide does not contain a consensus site for cAMP-dependent protein kinase A (although such a site is present elsewhere on the molecule and apparently unused), suggesting that the signaling cascade following intracellular cAMP production is more complex than originally thought. (ii) Characterizing the functional form of the cotransporter. During the present reporting period we have shown that the cotransporter exists as a homodimer in the acinar cell membrane. Briefly stated, this was established by demonstrating that in both intact and Triton-X100 solubilized membranes the cotransporter can be chemically crosslinked to another protein of the same molecular weight. Using a variety of experimental strategies we have demonstrated that this """"""""other"""""""" protein is another cotransporter molecule. (iii) Use of heterologous expression systems in order to obtain sufficient quantities of functional cotransporter protein for future structure/function studies. Our initial experiments during the present reporting period indicate that the yeast, S. cerevisiae, is capable of expressing the rat secretory Na-K-2Cl cotransporter at levels comparable to many mammalian tissues.
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