Function of SNAP-23 in H+-ATPase Trafficking in the IMCD

描述(申请人提供)：内髓集合管是非挥发性酸/碱的主要位置。IMCD还负责水通道蛋白-2(AQP-2)介导的尿水重吸收。质子泵(H+-ATPase)和AQP-2都是IMCD细胞的顶端靶点。在这种胞吐作用模型中，含有H+-ATPase或AQP2的囊泡表面的一种蛋白(S)与其同源蛋白膜蛋白发生特异性结合，称为v-SNARE(VAMP)，称为t-SNARE(Synaxin)。这些蛋白质相互作用形成7S复合体，然后与胞质蛋白相互作用，NSF和SNAP形成20S融合复合体。这两个过程都受到质子泵或AQP-2通道胞外插入和内吞回收平衡的调节。 长期目标是阐明赋予蛋白质运输特异性的机制-哪些蛋白质负责AQP-2和H+-ATPase的不同调控。此外，普遍存在的蛋白SNAP-23的作用尚未阐明。利用IMCD细胞系的培养，将研究SNAP-23的作用。GFP连接的SNAP-23将在IMCD细胞系中过表达。与野生型IMCD细胞相比，它们对不同pH挑战的反应将通过定量mRNA分析、蛋白质印迹分析、免疫细胞化学技术和共聚焦显微镜来表征。此外，还将研究Synaxin IA和SNAP-23的运输和结合特征。

DESCRIPTION (provided by applicant): The inner medullary collecting duct is a major location of non-volatile acid/base. The IMCD is also responsible for aquaporin- 2 (AQP-2) mediated water reabsorption from the urine. Both the proton pump (H+-ATPase) and AQP-2 are apically targeted in the IMCD cells. In this model of exocytosis, the specific association of a protein(s) on the surface of an H+-ATPase or AQP2 containing vesicle termed v-SNARE (VAMP), and its cognate partner, a membrane protein, termed t-SNARE (Syntaxin). These proteins interact to form a 7S complex, and then, with the interaction of the cytosolic proteins, NSF and SNAP form a 20S fusion complex. Both processes are rate regulated by the balance of exocytic insertion and endocytic retrieval of either the proton pump or the AQP-2 channel. The long term objectives are to elucidate the mechanisms which confer specificity to protein trafficking - which proteins are responsible for the differential regulation of AQP-2 versus H+-ATPase. Further, the role of the ubiquitous protein SNAP-23 has not been elucidated. Utilizing IMCD cell lines in culture, the role of SNAP-23 will be investigated. GFP attached SNAP-23 will be overexpressed in an IMCD cell line. Their response to different pH challenges, compared to wild-type IMCD cells, will be characterized by quantitative mRNA analysis, western blot analysis, immunocytochemical techniques and confocal microscopy. Further, Syntaxin IA and SNAP-23 trafficking and binding characteristics will be examined.