Salt Transport in Surgical Diarrheal Disease

描述(由申请人提供)： 从囊性纤维化到分泌性腹泻，上皮分泌调节缺陷是各种疾病的基础。本项目已经确定了基底外侧转运体(Na-K-2CI共转运体NKCC1)在控制上皮性CI分泌中的重要性，并确定了一种新的调节机制，涉及蛋白激酶C(PKC)介导的NKCC1表面表达的改变。现在建议进行实验，以绘制NKCC1的内吞途径，并描述特定的PKC亚型在NKCC1内化中的作用。新的数据表明，PKC介导的NKCC1的胞内内化(以及可能的Na-HCO3共转运体NBC1的胞外插入)可能在一定程度上解释了CI和HCO3在各种分泌上皮细胞的转运之间的相互关系。Rho家族GTPase成员在PKC引导的内吞作用和膜/细胞骨架重塑中的潜在作用将被探索。介导NKCC1内化的新的PKC亚型在扩散和迁移过程中似乎也在片状脂膜的形成和细胞延伸中发挥作用。推测，这种新的PKC的一般作用可能代表了一种快速将基侧离子转运体重新部署到或远离上皮细胞前沿的方法，这是最近认识到的在上皮细胞迁移和伤口闭合中重要的特征。这些研究利用上皮细胞系、生化和遗传技术以及共聚焦显微镜来解决这几个问题。在目标1中，将研究NKCC1表面动力学对选定的PKC激活剂和抑制剂的响应，以及NBC1表面动力学将在钙、v2和二氧化碳刺激的HCO3分泌过程中被检测。在目标2中，NKCC1的内吞途径将使用功能和形态标准来定义，辅以EGFP-NKCC1构建。NKCC1中的内吞信号和决定内吞NKCC1命运的因素将被研究。最后，我们将研究NKCC1和NBC1在上皮扩散时的胞内重新分布，以及PKCs、PKCS和Rho家族GTPase靶标在相关的膜-细胞骨架重塑中的可能作用。新的PKC同工酶对质膜组成和可塑性的调节形成了一条共同的线索，将这些关于基底侧盐转运蛋白内吞调节的研究与一条涉及上皮修复过程中的膜动力学的新兴研究线联系在一起。

DESCRIPTION (provided by applicant): Defective regulation of epithelial secretion underlies diseases ranging from cystic fibrosis to secretory diarrhea. This project has established the importance of a basolateral transporter (the Na-K-2CI cotransporter NKCC1 ) in the control of epithelial CI secretion and identified a novel regulatory mechanism involving alterations in NKCC1 surface expression mediated by protein kinase C (PKC). Experiments are now proposed to map endocytic pathways of NKCC1 and to delineate the role of specific PKC isoforms in NKCC1 internalization. New data suggest that PKC-mediated endocytic internalization of NKCC1 (and, possibly, exocytic insertion of the Na-HCO3 cotransporter NBC1) may account in part for the reciprocal relationship between CI and HCO3 transport in various secretory epithelia. The potential role of Rho-family GTPase members in PKC-directed endocytosis, and in membrane/cytoskeletal remodeling will be explored. The novel PKC isoforms that mediate NKCC1 internalization also appear to play a role in lamellipodia formation and cell extension during spreading and migration. Speculatively, this general action of novel PKCs may represent a means to rapidly redeploy basolateral ion transporters to, or away from, the leading edge of epithelial cells, a recently-recognized characteristic important in epithelial cell migration and wound closure. These studies utilize epithelial cell lines, biochemical and genetic techniques, and confocal microscopy to address these several issues. In Aim 1, NKCC1 surface dynamics will be examined in response to selected PKC activators and inhibitors, and NBC1 surface dynamics will be examined during Ca v2- and CO2-stimulated HCO3 secretion. In Aim 2, the endocytic pathway of NKCC1 will be defined using functional and morphologic criteria, aided by the use of an EGFP-NKCC1 construct. Endocytosis signals in NKCC1 and the factors that determine the fate of endocytosed NKCC1 will be examined. Finally, the endocytic redistribution of NKCC1 and NBC1 in response to epithelial spreading, and the possible role of PKCs, PKCS, and Rho-family GTPase targets in the associated membrane-cytoskeletal remodeling, will be examined. The regulation of plasma membrane composition and plasticity by novel PKC isoenzymes forms a common thread linking these studies of the endocytic regulation of basolateral salt transporters to an emerging line of investigation involving membrane dynamics during epithelial repair.