Irbit mediates synergy between Ca²⁺ and cAMP signaling pathways during epithelial transport in mice

Background & Aims: The cyclic adenosine monophosphate (cAMP) and Ca²⁺ signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. Methods: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6^-/- mice) and inositol 1,4,5-triphosphate (InsP₃) receptor-binding protein released with InsP₃ (Irbit^-/-) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl^- current. Protein interactions were determined by immunoprecipitation analyses. Results: Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca²⁺ and cAMP. In resting cells, Irbit was sequestered by InsP₃ receptors (IP ₃Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP₃Rs, which increased their affinity for InsP₃ and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP₃, caused dissociation of Irbit from IP₃Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit ^-/- or Slc26a6^-/- mice, or in salivary gland ducts expressing mutant forms of IP₃Rs that could not undergo protein kinase A-mediated phosphorylation. Conclusions: Irbit promotes synergy between the Ca²⁺ and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.