Vacuolar-type H + -ATPases (V-ATPases) contribute to pH regulation and play key roles in secretory and endocytic pathways. Dense-core vesicles (DCVs) in neuroendocrine cells are maintained at an acidic pH, which is part of the electrochemical driving force for neurotransmitter loading and is required for hormonal propeptide processing. Genetic loss of CAPS1 (aka calcium-dependent activator protein for secretion, CADPS), a vesicle-bound priming factor required for DCV exocytosis, dissipates the pH gradient across DCV membranes and reduces neurotransmitter loading. However, the basis for CAPS1 binding to DCVs and for its regulation of vesicle pH has not been determined. Here, MS analysis of CAPS1 immunoprecipitates from brain membrane fractions revealed that CAPS1 associates with a rabconnectin3 (Rbcn3) complex comprising Dmx-like 2 (DMXL2) and WD repeat domain 7 (WDR7) proteins. Using immunofluorescence microscopy, we found that Rbcn3α/DMXL2 and Rbcn3β/WDR7 colocalize with CAPS1 on DCVs in human neuroendocrine (BON) cells. The shRNA-mediated knockdown of Rbcn3β/WDR7 redistributed CAPS1 from DCVs to the cytosol, indicating that Rbcn3β/WDR7 is essential for optimal DCV localization of CAPS1. Moreover, cell-free experiments revealed direct binding of CAPS1 to Rbcn3β/WDR7, and cell assays indicated that Rbcn3β/WDR7 recruits soluble CAPS1 to membranes. As anticipated by the reported association of Rbcn3 with V-ATPase, we found that knocking down CAPS1, Rbcn3α, or Rbcn3β in neuroendocrine cells impaired rates of DCV reacidification. These findings reveal a basis for CAPS1 binding to DCVs and for CAPS1 regulation of V-ATPase activity via Rbcn3β/WDR7 interactions.

γ-Aminobutyric acid type A (GABAA) receptor plasticity participates in mediating adaptation to environmental change. Previous studies in rats demonstrated that extrasynaptic GABAA receptor subunits and receptors in the pons, a brainstem region involved in respiratory control, are upregulated by exposure to sustained hypobaric hypoxia. In these animals, expression of the mRNA encoding the extrasynaptic α4 subunit rose after 3 days in sustained hypoxia, while those encoding the α6 and δ subunits increased dramatically by 2 weeks. However, the participation of extrasynaptic subunits in maintaining respiration in normoxic conditions remains unknown. To examine the importance of α4 in a normal environment, respiratory function, motor and anxiety-like behaviors, and expression of other GABAA receptor subunit mRNAs were compared in wild-type (WT) and α4 subunit-deficient mice. Loss of the α4 subunit did not impact frequency, but did lead to reduced ventilatory pattern variability. In addition, mice lacking the subunit exhibited increased anxiety-like behavior. Finally, α4 subunit loss resulted in reduced expression of other extrasynaptic (α6 and δ) subunit mRNAs in the pons without altering those encoding the most prominent synaptic subunits. These findings on subunit-deficient mice maintained in normoxia, in conjunction with earlier findings on animals maintained in chronic hypoxia, suggest that the expression and regulation of extrasynaptic GABAA receptor subunits in the pons is interdependent and that their levels influence respiratory control as well as adaptation to stress.

Ca2+-dependent secretory granule fusion with the plasma membrane is the final step for the exocytic release of inflammatory mediators, neuropeptides, and peptide hormones. Secretory cells use a similar protein machinery at late steps in the regulated secretory pathway, employing protein isoforms from the Rab, Sec1/Munc18, Munc13/CAPS, SNARE, and synaptotagmin protein families. However, no small-molecule inhibitors of secretory granule exocytosis that target these proteins are currently available but could have clinical utility. Here we utilized a high-throughput screen of a 25,000-compound library that identified 129 small-molecule inhibitors of Ca2+-triggered secretory granule exocytosis in RBL-2H3 mast cells. These inhibitors broadly fell into six different chemical classes, and follow-up permeable cell and liposome fusion assays identified the target for one class of these inhibitors. A family of 2-aminobenzothiazoles (termed benzothiazole exocytosis inhibitors or bexins) was found to inhibit mast cell secretory granule fusion by acting on a Ca2+-dependent, C2 domain–containing priming factor, Munc13-4. Our findings further indicated that bexins interfere with Munc13-4–membrane interactions and thereby inhibit Munc13-4–dependent membrane fusion. We conclude that bexins represent a class of specific secretory pathway inhibitors with potential as therapeutic agents.