-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...
Recycling of H(+)-ATPase to the apical plasma membrane, mediated by vesicular exocytosis and endocytosis, is an important mechanism for controlling H(+) secretion by the collecting duct. We hypothesized that SNAREs (soluble N-ethylmaleimide-sensitive factor attachment proteins) may be involved in the targeting of H(+)-ATPase-coated vesicles. Using a tissue culture model of collecting duct H(+) secretory cells (inner medullary collecting duct (IMCD) cells), we demonstrated that they express the proteins required for SNARE-mediated exocytosis and form SNARE-fusion complexes upon stimulation of H(+)-ATPase exocytosis. Furthermore, exocytic amplification of apical H(+)-ATPase is sensitive to clostridial toxins that cleave SNAREs and thereby inhibit secretion. Thus, SNAREs are critical for H(+)-ATPase cycling to the plasma membrane. The process in IMCD cells has a feature distinct from that of neuronal cells: the SNARE complex includes and requires the vesicular cargo (H(+)-ATPase) for targeting. Using chimeras and truncations of syntaxin 1, we demonstrated that there is a specific cassette within the syntaxin 1 H3 domain that mediates binding of the SNAREs and a second distinct H3 region that binds H(+)-ATPase. Utilizing point mutations of the B1 subunit of the H(+)-ATPase, we document that this subunit contains specific targeting information for the H(+)-ATPase itself. In addition, we found that Munc-18-2, a regulator of exocytosis, plays a multifunctional role in this system: it regulates SNARE complex formation and the affinity of syntaxin 1 for H(+)-ATPase.
The authors describe a variant of Noonan's syndrome hitherto undescribed. The special findings in this case were anomalous origin of the aortic arch vessels and focal glomerulonephritis.
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