-Exocytic insertion of Hϩ -ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H ϩ -ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H ϩ -ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 Ϯ 7% and increased the interaction between GFP-syntaxin 1A and H ϩ -ATPase by 170 Ϯ 23%. Apical membrane Munc-18-2 decreased by 27.5 Ϯ 4.6% and H ϩ -ATPase increased by 246 Ϯ 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H ϩ -ATPase. In a pull-down assay of H ϩ -ATPase by glutathione S-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H ϩ -ATPase pulled down by 64 Ϯ 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H ϩ -ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H ϩ -ATPase, synaptosomeassociated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H ϩ -ATPase vesicles.soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; cell pH; acid secretion THE SOLUBLE N-ETHYLMALEIMIDE-SENSITIVE factor (NSF) attachment protein target receptor (SNARE) hypothesis of vesicle transport postulates that proteins in vesicular and target membranes form a core complex that leads to the docking and fusion of these membranes. Synaptobrevin or vesicle-associated membrane proteins (VAMPs) are primarily located on vesicles, and syntaxins are located on the target membranes. VAMP and syntaxin along with synaptosome-associated protein 23 or 25 (SNAP-23, the isoform expressed in somatic tissue, or SNAP-25, the isoform expressed in neuroexcitable tissue) form a parallel four-helix bundle. VAMP and syntaxin each contribute one helix and SNAP-23 or -25 contributes two helices (16) to form the four-helix fusion complex. The formation of this complex is thought to provide the energy required to overcome the energetic barrier of lipid bilayer fusion (16). Syntaxin has been found to have three different conformational arrangements: one in isolation, another when bound to Munc, and a third as part of the core SNARE complex (16). It has been proposed that when syntaxin is bound to Munc, it is in a "closed conformation," which does not allow...