Renal Epithelial Sodium Channels and Syntaxins

在致密上皮中，产电钠重吸收由位于顶膜的钠选择性阿米洛利敏感通道（ENaC）介导。 调节机制包括单通道水平的动力学效应、蛋白质合成和蛋白质从细胞内库向细胞表面的募集。 突触融合蛋白介导囊泡运输并且是NSF-SNAP-SNARE融合复合物的成员。 最近的数据表明，突触融合蛋白相互作用，并在功能上调节一些离子通道和泵。 突触融合蛋白亚型增加ENaC通道复合物的净外化，但功能调节是亚型特异性的（突触融合蛋白1A抑制，而突触融合蛋白3刺激阿米洛利敏感电流时，在非洲爪蟾卵母细胞中表达）。 据推测，突触融合蛋白影响ENaC的表达和活性取决于突触融合蛋白和ENaC亚基的功能结构域。 mpkCCDC 14细胞系是源自小鼠主细胞的生理学相关系统，其具有ENaC表达，并对醛固酮产生阿米洛利敏感性电流增加的响应。突触融合蛋白1a、3和4以及SNARE蛋白（SNAP 25和Munc 18）很容易检测到;共聚焦图像显示突触融合蛋白在顶膜中表达。 用反义寡核苷酸获得的数据表明突触融合蛋白在调节ENaC功能中的重要作用。 具体目标包括：1）、目的：研究突触融合蛋白及其辅助蛋白和其他SNARE在小鼠集合管主细胞系mpkCCDC 14中的表达。 2）。目的：研究mpkCCDC 14细胞中特异性Syntaxins和SNARE蛋白与ENaC复合物的相互作用。 3）。了解Syntaxin 1A和Syntaxin 3在卵母细胞表达系统和mpkCCDC 14细胞中对ENaC活性和表达的差异。 4）。利用非洲爪蟾卵母细胞表达系统和mpkCCDC 14细胞，确定小鼠ENaC亚基和syntaxin亚型的相互作用结构域和SNARES。 5）。研究醛固酮对mpkCCDC 14细胞中Syntaxins和SNARES表达的调控。 本提案将使用多种方法，包括电压钳测量和单通道测量、免疫沉淀、共聚焦显微镜和代谢标记。 表位作图将用于优化多克隆抗体的特异性，反义寡核苷酸将用于调节mpkCCDC 14系中内源性蛋白质的水平。

In tight epithelia, electrogenic sodium reabsorption is mediated by a sodium-selective amiloride-sensitive channel (ENaC), located in the apical membrane. Regulatory mechanisms include kinetic effects at the single channel level, protein synthesis and recruitment of proteins to the cell surface from intracellular pools. Syntaxins mediate vesicle trafficking and are members of the NSF-SNAP-SNAREs fusion complex. Recent data suggest that the syntaxins interact with and functionally regulate a number of ion channels and pumps. Syntaxin isoforms increase the net externalization of the ENaC channel complex, but functional regulation is isoform-specific (syntaxin 1A inhibits while syntaxin 3 stimulates the amiloride-sensitive currents when expressed in Xenopus oocytes). It is hypothesized that syntaxins affect ENaC expression and activity depending on the functional domains of the syntaxins and the ENaC subunits. The mpkCCDC14 cell line is a physiologically relevant system derived from mouse principal cells which has ENaC expression, and responds to aldosterone with an increase in amiloride-sensitive currents. Syntaxin 1a, 3 and 4 and SNARE proteins (SNAP25 and Munc18) are readily detectable; confocal images show the syntaxins to be expressed in the apical membrane. Data obtained with anti-sense oligonucleotides suggest an important role for syntaxins in regulating ENaC function. The specific aims include: 1): To define the physical expression of syntaxins, their accessory proteins and other SNARE in a Novel Immortalized Mouse Collecting Duct Principal Cell Line (mpkCCDC14). 2). To define the functional interactions between specific Syntaxins and other SNARE proteins with the ENaC Complex in mpkCCDC14cells. 3). To understand the differences between Syntaxin 1A and Syntaxin 3 on ENaC activity and expression in the oocyte expression system and in mpkCCDC14 cells. 4). To define the interacting domains of mouse ENaC subunits and syntaxin isoforms and the SNARES, using the Xenopus oocyte expression system and mpkCCDC14 cells. 5). To study the regulation of ENaC by aldosterone in the context of Syntaxins and SNARES expressed in mpkCCDC14 cells. A variety of methods will be used in this proposal, including voltage clamp measurements and single channel measurements, immunoprecipitation, confocal microscopy, and metabolic labeling will be utilized. Epitope mapping will be used to optimize the specificity of polyclonal antibodies, and anti-sense oligonucleotides will be used to modulate the level of endogenous proteins in the mpkCCDC14 line.