Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells.
Exocytic insertion of H(+)-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(+)-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(+)-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 +/- 7% and increased the interaction between GFP-syntaxin 1A and H(+)-ATPase by 170 +/- 23%. Apical membrane Munc-18-2 decreased by 27.5 +/- 4.6% and H(+)-ATPase increased by 246 +/- 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(+)-ATPase. In a pull-down assay of H(+)-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(+)-ATPase pulled down by 64 +/- 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(+)-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H(+)-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H(+)-ATPase vesicles.
Nicoletta
Julie A
JA
Renl Section, Boston University Medical Center, Boston, Massachusetts 02118, USA.
Ross
Jonathan J
JJ
Li
Guangmu
G
Cheng
Qingzhang
Q
Schwartz
Jonathon
J
Alexander
Edward A
EA
Schwartz
John H
JH
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007679:Kidney Medulla / Q000187:drug effects / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000378:metabolism*; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins / Q000187:drug effects / Q000378:metabolism*
D000954:Antigens, Surface; D004791:Enzyme Inhibitors; D051938:Munc18 Proteins; D009419:Nerve Tissue Proteins; C494213:Stx1a protein, rat; C497942:Stxbp2 protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D011493:Protein Kinase C; D000251:Adenosine Triphosphatases