Regulation of hIK1 in Secretory Diarrhea

描述（由申请人提供）：钙介导的激动剂是单独作用的肠分泌性腹泻的重要调节剂（例如，轮状病毒，副溶血弧菌）或与cAMP介导的激动剂协同作用。事实上，美国每年的医疗费用估计为230亿美元。Cl-分泌过程中的关键步骤是基底外侧膜Ca 2+激活的K+通道hlK1的激活。由于跨上皮Cl-分泌本质上是载体性的，因此离子转运蛋白和通道正确靶向顶膜或基底外侧膜至关重要。到目前为止，还没有关于hlKl组装并运输到质膜（一般而言）和特别是基底外侧膜的机制的信息。我们的长期目标是确定负责正确组装，运输，基底侧定位和调节hIK1的分子基序。在具体目标A中，我们将定义C-末端亮氨酸拉链调节hlKl的折叠和运输以及调节的机制。此外，我们表明，以下的N-末端亮氨酸拉链突变，hIK1未能交通质膜。在特定目标B中，我们将确定N-末端是否作为四聚化结构域，N-末端是否与C-末端相互作用，以及突变是否影响通道调控。此外，我们将确定hlK1是否与KCNN基因家族的其他成员组装成异源四聚体，以及确定这种共组装的生理和药理学后果。在特定目标C中，我们将定义hIK1在分化上皮中基底外侧膜定位所需的分子基序。对于这些研究，我们将使用MDCK细胞系。我们将确定一般的hlK1和上皮特异性mu1B的本地化衔接子的作用。最后，我们将确定hIK1内吞的速率，并确定卡巴胆碱，胰岛素和EGF是否调节这一过程。我们所有的研究将涉及膜片钳，蛋白质生化和免疫荧光技术的组合HEK293，LLC-PK1和MDCK细胞异源表达hIK1。总的来说，我们的研究将首次定义KCNN基因家族成员正确组装、运输和定位所需的分子基序。由于hIK1在Ca2+介导的分泌性腹泻中起着关键作用，并且可能是CF表型的修饰基因，因此对hIK1的运输和调节的理解将有望在临床上有用

DESCRIPTION (provided by applicant): Calcium-mediated agonists are important modulators of intestinal secretory diarrhea acting alone (e.g., rotavirus, Vibrio parahaemolyticus) or in synergism with cAMP-mediated agonists. Indeed, medical costs are estimated at $23 billion annually in the United States. A critical step in the Cl- secretory process is the activation of a basolateral membrane Ca2+-activated K+ channel, hlK1. As transepithelial Cl- secretion is vectorial in nature it is critical that the ion transporters and channels be correctly targeted to either the apical or basolateral membrane. To date, there is no information regarding the mechanisms by which hlK1 is assembled and trafficked to the plasma membrane in general and to the basolateral membrane in particular. Our long-term goals are to define the molecular motifs responsible for the correct assembly, trafficking, basolateral localization and regulation of hIK1. In Specific Aim A, we will define the mechanism by which the C-terminal leucine zipper regulates folding and trafficking and regulation of hlK1. Also, we demonstrate that following mutation of the N-terminal leucine zipper, hIK1 fails to traffic to the plasma membrane. In Specific Aim B we will determine whether the N-terminus acts as a tetramerization domain, whether the N-terminus interacts with the C-terminus and whether mutations affect channel regulation. Also, we will determine whether hlK1 assembles into heterotetramers with other members of the KCNN gene family as well as determine the physiological and pharmacological consequences of this co-assembly. In Specific Aim C we will define the molecular motifs required for basolateral membrane localization of hIK1 in differentiated epithelia. For these studies we will utilize the MDCK cell line. We will determine the role of adaptors in the localization of hlK1 in general and the epithelial specific mu1B in particular. Finally, we will define the rate of hIK1 endocytosis and determine whether carbachol, insulin and EGF regulate this process. All of our studies will involve a combination of patch-clamp, protein biochemical and immunofluorescence techniques on HEK293, LLC-PK1 and MDCK cells heterologously expressing hIK1. In total, our studies will define, for the first time, the molecular motifs required for the correct assembly, trafficking and localization of a member of the KCNN gene family. As hIK1 plays a critical role in Ca2+-mediated secretory diarrhea and may be a modifier gene of the CF phenotype an understanding of the trafficking and regulation of hIK1 would be expected to be clinically useful