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1 Medicine/Renal, Physiology, Pathology, Boston University School of Medicine, Boston, MA, USA
* To whom correspondence should be addressed. E-mail: jhsch{at}bu.edu.
Exocytic insertion of H+-ATPase into the apical plasma membrane of inner medullary collecting duct (IMCD) cells is stimulated by cell acidification and is dependent upon formation of a SNARE complex. Munc 18 regulates SNARE complex formation. The purpose of these studies was to determine the role of Munc 18 in the exocytosis of H+-ATPase in the IMCD cell. We compared the effect of acute cell acidification of IMCD cells, transfected with GFP- Syntaxin 1A, on the interaction of Syntaxin 1A with Munc 18-2 and the 31 kD subunit of the H+-ATPase. Immunoprecipitation studies revealed that cell acidification decreased GFP-Syntaxin 1A and Munc 18-2 interaction by 49 ± 7% while the interaction between GFP-Syntaxin 1A and H+-ATPase increased by 170 ±23%. Munc 18-2 in the apical membrane decreased by 27.5 ± 4.6%, the 31 kD subunit of the H+-ATPase increased by 246± 22% while an apical membrane marker, GP-135 did not change after cell acidification. Pretreatment of IMCD cells with a PKC inhibitor (G0 6983) diminished the prior described changes in Munc 18-2 syntaxin 1A interaction and redistribution of H+-ATPase with cell acidification. In a pull down assay of H+-ATPase by GST- Syntaxin 1A bound to sepharose beads, preincubation of the beads with His-Munc 18-2 decreased the amount of H+-ATPase pulled down. A ~2 fold excess of Munc 18-2 reduced H+-ATPase binding by 64 ±16%. Preincubating the beads with an equivalent amount of albumin had no effect on the amount of H+-ATPase pulled down. IMCD transfected to overexpress Munc 18-2 had a reduced rate of proton transport compared to control cells. From these studies, we conclude that Munc 18-2 plays an important role in the regulation of H+-ATPase exocytosis. We hypothesize that Munc 18-2 must dissociate from the Syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in Syntaxin 1A, allowing it to interact with H+-ATPase, SNAP- 23 and VAMP, forming the SNARE complex that leads to the docking and fusion of vesicles containing H+-ATPase.
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