PROJECT SUMMARY / ABSTRACT During enamel formation (amelogenesis) the regulation of extracellular pH (pHe) is critical as shown by multiple mutant mice lines. Bicarbonate (HCO3-) export to the enamel matrix involves anion exchanger 2 (AE2) and members of the SLC26A gene family. AE2 is localized to the lateral membrane of maturation ameloblasts, while SLC26A1, 3, 4, 6 and 7 all localize to the apical/distal membrane of these same cells. In maturation ameloblasts SLC26A proteins colocalize with the cystic fibrosis transmembrane conductance regulator (CFTR) and function together to allow for the export of HCO3- and the bidirectional movements of chloride ions (Cl-) as part of the pHe regulatory process. In other polarized epithelial cell types of a secretory nature, SLC26A proteins and CFTR form a network with cytoskeletal filamentous actin (F-actin) at the apical pole dictating, to a large part, cell polarity and microvilli projections. NHERF1/SLC9A3R1 and Ezrin proteins are also involved with this network interaction. The pH-sensitive sodium dependent phosphate transporter (SLC34A2/NaPi2b), an inorganic phosphate (Pi) import channel also localizes to the apical pole of maturation ameloblasts, which may suggest a direct link between pHe regulation and Pi transport activities during enamel mineralization events. Data suggests that miRNAs, targeting the mRNAs of specific ion transport proteins, also influence ion transport and pHe regulation. Our prior whole transcriptome analysis of maturation and secretory enamel organ cells identify a potential role for miR-298 and miR-346 in the regulation of NHERF1, Slc26a1, Slc26a7 and Cftr. In this application we hypothesize that a “SLC26A/CFTR/NHERF1/NaPi2b network, directing ion movements directly related to HCO3- and Pi transport and pH regulation, while at the same time dictating apical membrane architecture, is critical for enamel maturation, and disruptions to this network result in enamel pathologies that will severely impact on enamel longevity.” We propose the following specific aims: 1) immunolocalization of NHERF1, EZRIN, CFTR and NaPi2b in maturation ameloblasts; 2) disrupt SLC26A/CFTR/NHERF1/NaPi2b network in enamel organ cells in vivo using miR-346 and miR-298 delivered directly at the site of mineralization; and; 3) investigate the nature of the NHERF1 and NaPi2b interactions in enamel organ cells in vitro and in vivo. In this study we anticipate that disrupting the SLC26A/CFTR network and NaPi2b activity, by directly targeting NHERF1 function, will result in a significant enamel dysmorphology. Findings from this study will have a significant impact on our understanding of the biomineralization process as it relates to enamel formation in all mammalian species. This study may also lead to strategies for handling inherited enamel defects in the clinic; and, in the long run help to prevent and alleviate the suffering of those afflicted with cystic fibrosis and other diseases.