Renal Epithelial Sodium Channels and Syntaxins

EXCEEDTHE SPACEPROVIDED。在致密上皮中，电致钠重吸收是由位于顶端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。Syntaxins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。Syntaxin异构体增加ENaC通道复合物的净外化，但功能调节是异构体特异性的（Syntaxin 1A抑制而Syntaxin 3在爪蟾卵母细胞中表达时刺激阿米洛利敏感电流）。据推测，句法素影响ENaC的表达和活性取决于句法素和ENaC亚基的功能域。mpkCCDcl4细胞系是源于小鼠主细胞的生理相关系统，具有ENaC表达，并对醛固酮有反应，增加酰胺敏感电流。Syntaxin 1a、3、4和SNARE蛋白（SNAP25和Munc18）很容易检测到；共聚焦图像显示合成素在顶膜中表达。反义寡核苷酸的数据表明，合成素在调节ENaC功能中起着重要作用。具体目的包括：1)：确定合成素及其附属蛋白和其他SNARE在新型永生化小鼠收集管主细胞系（mpkCCDc14）中的物理表达。2）. 定义mpkccdc14细胞中特定Syntaxins和其他SNARE蛋白与ENaC复合物之间的功能相互作用。3)。了解Syntaxin 1A和Syntaxin 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)。利用爪蟾卵母细胞表达系统和mpkCCDc- bbb4细胞，确定小鼠ENaC亚基和syntaxin亚型与SNAREs的相互作用域。5)。研究醛固酮在mpkCCDc- bbbb4细胞Syntaxins和SNAREs表达背景下对ENaC的调控作用。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mpkCCDc-|4 line PERFORMANCE SITE ========================================Section End===========================================中的内源性蛋白水平

EXCEEDTHE SPACEPROVIDED. 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 mpkCCDcl4 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 mpkCCDc-|4 cells. 5). To study the regulation of ENaC by aldosterone in the context of Syntaxins and SNAREs expressed in mpkCCDc-|4 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 mpkCCDc-|4 line PERFORMANCE SITE ========================================Section End===========================================