The major objective of the proposed research is to characterize the molecular mechanisms of chloride/anion transporters in the basolateral and apical membranes in the human intestine. Recent studies have demonstrated a family of Cl--HCO3- anion exchangers (AE) represented by AE1, AE2, brain AE3, and cardiac AE3. AE2 and bAE3 are localized to the basolateral membranes of human intestine. The applicant hypothesizes that (1) the two basolateral membrane AE isoforms, AE2 and bAE3 (but not AE1 and cAE3) may be differentiall expressed and regulated, and (2) that the human apical membrane isoform remain to be identified and cloned. The proposed specific aims will address (a) the vertical and regional localization of AE2 and bAE3, (b) the functions of AE2 and bAE3 in control of intracellular pH, cell volume and Cl transport, (c) growth factor regulation and signalling of AE2 and bAE3, and (d) the cloning o full length cDNA of the putative apical membrane AE isoform. A variety of biochemical, imaging, and molecular biology techniques, including immunoblotting, in situ RT-PCR, immunohistochemistry, transient transfection, and 5' and 3' RACE will be utilized to address the aims. The results are expected to provide important insights into the molecular regulation of these intestinal anion exchangers in the human intestine which could provide a basis for understanding pathophysiological conditions, such as diarrhea disorders, associated with function of these proteins.
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