SNARE proteins regulate H+-ATPase redistribution to the apical membrane inrat renal inner medullary collecting duct cells

The interaction of soluble N-ethylmaleimide-sensitive factor attachment pro tein receptor (SNARE) proteins provides the necessary steps for vesicle doc king fusion, In inner medullary collecting duct (IMCD) cells, acid secretio n is regulated in part by exocytotic insertion and endocytotic retrieval of an H+-ATPase to and from the apical membrane. We previously suggested a ro le 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 ex ocytosis and to determine the effects of clostridial toxins on SNARE-mediat ed trafficking of H+-ATPase. Cell acidification induced a marked increment of H+-ATPase in the apical membrane. However, pretreating cells with clostr idial toxins blocked the cellular translocation of the 31-kDa subunit. Immu noprecipitation of IMCD cell homogenate, using antibodies against either th e 31-kDa subunit of H+-ATPase or vesicle-associated membrane protein-2, co- immunoprecipitated N-ethylmaleimide-sensitive factor, alpha-soluble NSF att achment protein (alpha-SNAP), synaptosome-associated protein-23, syntaxin, and vesicle-associated membrane protein-2. Pretreatment with clostridial to xin resulted in reduced co-immunoprecipitation of WC-ATPase and syntaxin. T hese experiments document, for the first time, a putative docking fusion co mplex in IMCD cells and a physical association of the H+-ATPase with the co mplex, The sensitivity to the action of clostridial toxin indicates the doc king-fusion complex is a part of the exocytotic mechanism of the proton pum p.