SNARE proteins regulate H(+)-ATPase redistribution to the apical membrane in rat renal inner medullary collecting duct cells.
The interaction of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins provides the necessary steps for vesicle docking fusion. In inner medullary collecting duct (IMCD) cells, acid secretion is regulated in part by exocytotic insertion and endocytotic retrieval of an H(+)-ATPase to and from the apical membrane. We previously suggested a role for SNARE proteins in exocytotic insertion of proton pumps in IMCD cells. The purpose of the present study was to determine whether SNARE proteins are associated with the 31-kDa subunit of H(+)-ATPase in IMCD cells during exocytosis and to determine the effects of clostridial toxins on SNARE-mediated trafficking of H(+)-ATPase. Cell acidification induced a marked increment of H(+)-ATPase in the apical membrane. However, pretreating cells with clostridial toxins blocked the cellular translocation of the 31-kDa subunit. Immunoprecipitation of IMCD cell homogenate, using antibodies against either the 31-kDa subunit of H(+)-ATPase or vesicle-associated membrane protein-2, co-immunoprecipitated N-ethylmaleimide-sensitive factor, alpha-soluble NSF attachment protein (alpha-SNAP), synaptosome-associated protein-23, syntaxin, and vesicle-associated membrane protein-2. Pretreatment with clostridial toxin resulted in reduced co-immunoprecipitation of H(+)-ATPase and syntaxin. These experiments document, for the first time, a putative docking fusion complex in IMCD cells and a physical association of the H(+)-ATPase with the complex. The sensitivity to the action of clostridial toxin indicates the docking-fusion complex is a part of the exocytotic mechanism of the proton pump.
Banerjee
A
A
Renal Section, Boston University Medical Center, Boston University School of Medicine, Boston, Massachusetts 02118-2908, USA.
Shih
T
T
Alexander
E A
EA
Schwartz
J H
JH
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D005089:Exocytosis; D007679:Kidney Medulla / Q000166:cytology / Q000201:enzymology*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000201:enzymology*; D008565:Membrane Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D051381:Rats; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins
D008565:Membrane Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D006180:Proton-Translocating ATPases