This proposal is directed at elucidating the role of the human specific Na+/H+ exchanger isoform, NHE-2, in Na+ absorption by the human intestinal tract. Studies have been designed specifically to examine the hypothesis that the relative contribution of the individual isoforms, NHE-2 or NHE-3, in Na+ absorption is region specific within the human intestinal tract, and that the two putative apical isoforms are differentially expressed and regulated. Our studies focus on isolating the full length cDNA and the genomic clone for human NHE-2 in order to elucidate its structure-function relationships in the human intestine. Using the partial human NHE-2 cDNA obtained in our laboratory, the full length cDNA will be isolated by screening the human colonic cDNA library and by the RACE method. The chromosomal localization of the human NHE-2 gene will be carried out to determine its exact location relative to NHE-1 and NHE-3. Potential regulatory consensus sequences and post-translational modification sites will be mapped and compared to other NHE isoforms. RNase protection assays will be carried out utilizing RNA extracted from pinch biopsies of the human intestinal tract to determine the regional distribution of the various NHE isoforms. Immunolocalization and in situ hybridization methods have been designed to assess the cellular distribution and membrane localization of the NHE-2 and NHE-3 isoforms. Using human NHE-2 transfected PS 120 fibroblasts, the relationship of various protein domains to amiloride binding, Na+ transport and regulation by various kinases and growth factors will be evaluated by transport methods. Deletion mutations and site-directed mutagenesis studies will be carried out to determine the specific regions of the NHE-2 gene involved in Na+ transport and its regulation. Cell-surface biotinylation, immunoblotting and immunofluorescence studies will be designed to assess the trafficking of the NHE-2 and NHE-3 isoforms in CaCo-2 cells. The specific sequences required for protein targeting to the precise membrane domain will be elucidated by the use of chimeras between the different domains of the NHE isoforms. Utilizing the partial fragment and the full length cDNA, the promoters and regulatory elements of the human NHE-2 will be identified using the Luciferase reporter gene system. DNA-protein interactions specific for cellular control of expression of the human NHE-2 isoform will be investigated by mobility shift and DNA footprinting analysis. These studies are designed to yield valuable information on the cis- and trans-acting elements involved in the regulation of NHE-2 expression. Our proposed studies of the NHE-2 and NHE-3 isoforms in the human gastrointestinal tract would yield valuable information on the mechanisms of regulation of intestinal Na+ absorption and may have direct relevance to an understanding of the pathophysiology of diarrheal disorders and other alterations of intestinal electrolyte transport.
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