Banerjee A, Li G, Alexander E A, Schwartz J H
Renal Section, Boston University Medical Center, Boston University School of Medicine, Boston, MA 02118, USA.
Am J Physiol Cell Physiol. 2001 Apr;280(4):C775-81. doi: 10.1152/ajpcell.2001.280.4.C775.
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.
H⁺-ATP酶囊泡向髓质内集合管（IMCD）细胞顶端膜的转运利用了一种类似于神经分泌细胞中所描述的机制，该机制涉及可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白。这些囊泡的调节性胞吐作用与SNARE复合体的形成有关。特异性切割靶标（t-）SNARE、 syntaxin-1或囊泡SNARE、囊泡相关膜蛋白-2的梭菌神经毒素，会减少SNARE复合体的形成、H⁺-ATP酶向顶端膜的转运，并抑制H⁺分泌。这些实验的目的是确定第二种t-SNARE，即可溶性N-乙基马来酰亚胺敏感因子附着蛋白（SNAP）-23（神经元SNAP-25的同源物）在H⁺-ATP酶囊泡调节性胞吐作用中的生理作用。我们的实验证明，25 - 50 nM肉毒杆菌毒素（Bot）A或E可切割大鼠SNAP-23，从而在60分钟内使免疫可检测的和（³⁵）S标记的SNAP-23减少>60%。向IMCD匀浆中添加25 nM BotE可减少可从匀浆中免疫沉淀的20 S样SNARE复合体的量。用BotE处理完整的IMCD单层细胞，可使转运至顶端膜的H⁺-ATP酶量比对照减少52±2%，并在急性细胞酸化后使H⁺分泌速率降低77±3%。我们得出结论，SNAP-23是肉毒杆菌毒素蛋白水解的底物，在这些肾上皮细胞中对H⁺-ATP酶胞吐作用和H⁺分泌的调节中起关键作用。
