Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells.
Munc-18-2 regulates exocytosis of H(+)-ATPase in rat inner medullary collecting duct cells.
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Munc-18-2 调节大鼠内髓集合管细胞中 H(+)-ATP 酶的胞吐作用。
DOI：
10.1152/ajpcell.00588.2003
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Schwartz,JohnH
中科院分区：
文献类型：
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作者：
Nicoletta,JulieA;Ross,JonathanJ;Li,Guangmu;Cheng,Qingzhang;Schwartz,Jonathon;Alexander,EdwardA;Schwartz,JohnH
胞外插入H+- atp酶进入内髓集管（IMCD）细胞的顶膜依赖于可溶性-乙基马来酰亚胺敏感因子（NSF）附着蛋白靶受体（SNARE）复合物。在这项研究中，我们确定了Munc-18在调节IMCD细胞H+- atp酶分泌中的作用。我们比较了急性细胞酸化（IMCD胞分泌的刺激）对syntaxin 1A与Munc-18-2和H+- atp酶的31 kda亚基的相互作用的影响。免疫沉淀显示，细胞酸化使绿色荧光蛋白(GFP)-syntaxin 1A与Munc-18-2的相互作用降低了49±7%，使GFP-syntaxin 1A与H+- atp酶的相互作用增加了170±23%。顶膜标志物Munc-18-2和H+- atp酶分别降低了27.5±4.6%和246±22%，而GP-135没有升高。用PKC抑制剂（GO-6983）预处理IMCD细胞，可以减少先前描述的Munc-18-2-syntaxin 1A相互作用和H+- atp酶重新分配的变化。在谷胱甘肽转移酶(GST)-syntaxin 1A结合珠粒的H+- atp酶拉降实验中，预孵卵的珠粒与His-Munc-18-2的大约两倍过量减少了H+- atp酶拉降64±16%。与对照细胞相比，过表达Munc-18-2的IMCD细胞质子转运率降低。我们得出结论，Munc-18-2必须与syntaxin 1A蛋白分离，才能发生H+- atp酶的胞外分泌。这种解离导致syntaxin 1A的构象改变，使其能够与H+- atp酶、突触体相关蛋白(SNAP)-23和囊泡相关膜蛋白（VAMP）相互作用，形成SNARE复合物，导致H+- atp酶囊泡的对接和融合。
Exocytic insertion of H+-ATPase into the apical membrane of inner medullary collecting duct (IMCD) cells is dependent on a solubleN-ethylmaleimide-sensitive factor (NSF) attachment protein target receptor (SNARE) complex. In this study we determined the role of Munc-18 in regulation of IMCD cell exocytosis of H+-ATPase. We compared the effect of acute cell acidification (the stimulus for IMCD exocytosis) on the interaction of syntaxin 1A with Munc-18-2 and the 31-kDa subunit of H+-ATPase. Immunoprecipitation revealed that cell acidification decreased green fluorescent protein (GFP)-syntaxin 1A and Munc-18-2 interaction by 49 ± 7% and increased the interaction between GFP-syntaxin 1A and H+-ATPase by 170 ± 23%. Apical membrane Munc-18-2 decreased by 27.5 ± 4.6% and H+-ATPase increased by 246 ± 22%, whereas GP-135, an apical membrane marker, did not increase. Pretreatment of IMCD cells with a PKC inhibitor (GO-6983) diminished the previously described changes in Munc-18-2-syntaxin 1A interaction and redistribution of H+-ATPase. In a pull-down assay of H+-ATPase by glutathioneS-transferase (GST)-syntaxin 1A bound to beads, preincubation of beads with an approximately twofold excess of His-Munc-18-2 decreased H+-ATPase pulled down by 64 ± 16%. IMCD cells that overexpress Munc-18-2 had a reduced rate of proton transport compared with control cells. We conclude that Munc-18-2 must dissociate from the syntaxin 1A protein for the exocytosis of H+-ATPase to occur. This dissociation leads to a conformational change in syntaxin 1A, allowing it to interact with H+-ATPase, synaptosome-associated protein (SNAP)-23, and vesicle-associated membrane protein (VAMP), forming the SNARE complex that leads to the docking and fusion of H+-ATPase vesicles.