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 medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize 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 document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and (35)S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase 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 conclude 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.
Renal Section, Boston University Medical Center, Boston University School of Medicine, Boston, MA 02118, USA.
A
A
Banerjee
G
G
Li
E A
EA
Alexander
J H
JH
Schwartz
D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A / Q000494:pharmacology; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D005089:Exocytosis / Q000502:physiology; D007679:Kidney Medulla / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000032:analysis; D009465:Neuromuscular Agents / Q000494:pharmacology; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011522:Protons; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009465:Neuromuscular Agents; D011522:Protons; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D050683:R-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; C494212:STX1A protein, human; D052067:Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D006180:Proton-Translocating ATPases; C096326:botulinum toxin type E