Renal Epithelial Sodium Channels and Syntaxins

在紧密上皮中，电生钠重吸收是由位于顶膜的钠选择性阿米洛利敏感通道(ENaC)介导的。调节机制包括单通道水平的动力学效应、蛋白质合成和从细胞内池向细胞表面募集蛋白质。Synaxins介导囊泡运输，是NSF-SNAP-SNARES融合复合体的成员。最近的数据表明，合成素与许多离子通道和泵相互作用并在功能上调节。Synaxin异构体增加ENaC通道复合体的净外部化，但功能调节是异构体特有的(在非洲爪哇卵母细胞中表达时，Synaxin 1A抑制而Synaxin 3刺激对阿米洛利敏感的电流)。假设合成素影响ENaC的表达和活性依赖于合成素和ENaC亚单位的功能结构域。MpkCCDC14细胞系是从小鼠主细胞分化而来的一个生理相关系统，具有ENaC的表达，对醛固酮的反应伴随着阿米洛利敏感电流的增加。突触素1a、3和4以及SNARE蛋白(SNAP25和Munc18)很容易被检测到；共聚焦图像显示突触素在顶膜中表达。用反义寡核苷酸获得的数据表明，Synaxins在调节ENaC功能中发挥着重要作用。本研究的具体目的包括：1)建立一种新的永生化小鼠集合管主细胞系(MpkCCDC14)，并研究其合成素及其附属蛋白和其他诱导物的物理表达。2)。研究mpkCCDC14细胞中特定的Synaxins和其他SNARE蛋白与ENaC复合体之间的功能相互作用。3)。目的：了解Synaxin 1A和Synaxin 3在卵母细胞表达系统和mpkCCDC14细胞中ENaC活性和表达的差异。4)。利用非洲爪哇卵母细胞表达系统和mpkCCDC14细胞，确定小鼠ENaC亚基、合成素异构体和SNARs的相互作用结构域。5)。目的：研究醛固酮对mpkCCDC14细胞表达的Synaxins和SNARs的调节作用。在这项提案中将使用多种方法，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。表位定位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mpkCCDC14系中内源蛋白的水平。

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.