Role of SNAP-23 in trafficking of H+-ATPase in cultured inner medullary collecting duct cells

The trafficking of H+-ATPase vesicles to the apical membrane of inner medul lary collecting duct (IMCD) cells utilizes a mechanism similar to that desc ribed in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocy tosis of these vesicles is associated with the formation of SNARE complexes . Clostridial neurotoxins that specifically cleave the target (t-) SNARE, s yntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, red uce SNARE complex formation, H+-ATPase translocation to the apical membrane , and inhibit H+ secretion. The purpose of these experiments was to charact erize the physiological role of a second t- SNARE, soluble N-ethylmaleimide -sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments do cument that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and t hereby reduces immunodetectable and S-35-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 2 0 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPa se translocated to the apical membrane by 52 +/- 2% of control and reduces the rate of H+ secretion by 77 +/- 3% after acute cell acidification. We co nclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.