== Immunolocalization was performed using the procedures described previously in details (38). these transporters by different mechanisms. These findings define IRBIT as a key coordinator of epithelial fluid and HCO3secretion and may have implications to all CFTR-expressing epithelia and to cystic fibrosis. == Introduction == The cardinal function of most secretory glands ductal systems is the secretion of a HCO3-rich fluid. Aberrant fluid and HCO3secretion leads to several diseases, including cystic fibrosis (1) and pancreatitis (2). The pancreatic duct epitomizes this function by secreting the bulk of the fluid in the pancreatic juice that contains about 140 mM HCO3and 20 mM Cl(3). Fluid and HCO3secretion requires HCO3entry at the basolateral membrane (BLM) and HCO3exit at the luminal membrane (LM). Basolateral HCO3entry at the pancreatic duct is mediated by the Na+-HCO3cotransporter, pNBC1 (46). Luminal HCO3secretion (efflux) is coupled to Clabsorption (influx) and is mediated by the coordinated function of the solute carrier family 26 (SLC26) transporters (SLC26Ts) and the CFTR (710). The exact roles of the SLC26Ts and CFTR in mediating Clabsorption and HCO3secretion are not well understood. The pancreatic SLC26Ts, SLC26, member 3 (SLC26A3) and SLC26A6, function as coupled Cl/HCO3exchangers (8,1113), with isoform specific stoichiometry (8,14). Most models assume that the SLC26Ts mediate the coupled Clabsorption and HCO3secretion (3,15). Indeed, deletion of Slc26a6 in mice inhibited stimulated fluid and HCO3secretion (16). Although it is clear that CFTR is essential for pancreatic fluid and HCO3secretion, its role in the secretion is less certain. The original model proposed that CFTR-mediated secretion of Clto the duct lumen mainly sustains the Cl/HCO3exchange (17). However, several studies showed that CFTR has a limited but significant HCO3permeability (18,19), which led to the suggestion that CFTR mediates the coupled Clabsorption and HCO3secretion. One way to address this important hypothesis is to examine directly the role of CFTR in Cltransport in the mouse pancreatic duct. The mouse duct mediates fluid and HCO3secretion in a mechanism similar to that in human and guinea pig ducts (3,20). However, the mouse duct also mediates HCO3-independent fluid secretion that requires basolateral Clinflux via the Na+-K+-2Clcotransporter isoform 1 NKCC1 (20) and luminal HC-030031 Clefflux by an unknown transporter. In the present work, we took advantage of this activity to demonstrate that the main role of CFTR in the pancreatic duct is Clsecretion into the lumen. An important question concerning the epithelia is Rabbit Polyclonal to SH3RF3 how events at the BLM and LM are coordinated to ensure fidelity of the secretory process. A potential coordinator of pancreatic fluid and HCO3secretion is the protein named IRBIT (inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3). IRBIT was discovered as a protein that HC-030031 binds to IP3receptors (IP3Rs) (21) and competes with IP3for binding to the IP3-binding domain of the IP3Rs to regulate Ca2+signaling (22). IRBIT has several domains (see below): aa 3943 form a protein phosphatase 1 (PP1) binding ligand, which is followed by PEST (aa 6592) and coiled-coil domains (aa 111138), and C terminus PSD-95/discs large/zona occludens-1 (PDZ) ligand (aa 527530) (23). The PEST domain has 11 serines that can be phosphorylated, and phosphorylation of several serines, including S68, is required for binding of IRBIT to the IP3Rs (22). However, in vivo only phosphorylation of S68 appears to be regulated by PP1 (23), which inhibits binding of IRBIT to the IP3Rs. Recently, it was discovered that IRBIT potently activates pNBC1 but not HC-030031 the kidney-specific HC-030031 isoform kNBC1 (24). IRBIT binds to the N-terminal 143 residues of pNBC1 that are not present in kNBC1. Accordingly, when expressed inXenopusoocytes, IRBIT activates pNBC1 but not kNBC1 (24). Phosphorylation of the PEST domain is required for IRBIT binding to and activation of pNBC1 (24). Regulation of pNBC1 by IRBIT suggests that IRBIT may regulate pancreatic fluid and HCO3secretion. Here we show that IRBIT regulates HCO3-dependent and, unexpectedly, HCO3-independent fluid secretion. This turned out to be due to a prominent activation of CFTR by IRBIT, with IRBIT reducing CFTR closed-duration time to increase channel open probability. Activation of.