Salt Transport in Surgical Diarrheal Disease

描述（由申请人提供）： 上皮分泌调节缺陷是从囊性纤维化到分泌性腹泻等疾病的基础。该项目已经确定了基底外侧转运蛋白（Na-K-2CI协同转运蛋白NKCC 1）在控制上皮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NKCC 1表面表达的改变。现在提出的实验地图NKCC 1的内吞途径，并描绘特定的PKC亚型在NKCC 1内化的作用。新的数据表明，PKC介导的内吞内化的NKCC 1（和，可能，外吞插入的Na-HCO 3协同转运蛋白NBC 1）可能部分占CI和HCO 3运输在各种分泌上皮细胞之间的相互关系。将探讨Rho家族GT3成员在PKC介导的内吞作用和膜/细胞骨架重塑中的潜在作用。新的PKC亚型，介导NKCC 1内化也似乎发挥了作用，在板状伪足的形成和细胞的扩展和迁移过程中的延伸。推测，这种新的蛋白激酶的一般行动可能代表了一种手段，快速重新部署基底外侧离子转运蛋白，或远离，上皮细胞的前缘，最近认识到的特征，重要的上皮细胞迁移和伤口闭合。这些研究利用上皮细胞系，生化和遗传技术，共聚焦显微镜来解决这几个问题。在目标1中，NKCC 1表面动力学将被检查在响应于选定的PKC激活剂和抑制剂，和NBC 1表面动力学将被检查在Ca v2和CO2刺激的HCO 3分泌。在目的2中，NKCC 1的内吞途径将使用功能和形态学标准来定义，并通过使用EGFP-NKCC 1构建体来辅助。将检查NKCC 1中的内吞信号和决定内吞NKCC 1命运的因素。最后，NKCC 1和NBC 1的内吞再分配响应上皮扩散，以及PKCs，PKCS和Rho家族GT3靶点在相关的膜细胞骨架重塑中的可能作用，将被检查。新型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，将基底外侧盐转运蛋白的内吞调节研究与上皮修复过程中涉及膜动力学的新兴研究联系起来。

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.