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（如轮状病毒、副溶血性弧菌）或与camp介导的激动剂协同作用的肠分泌性腹泻的重要调节剂。事实上，美国每年的医疗费用估计为230亿美元。Cl-分泌过程中的一个关键步骤是激活基侧膜Ca2+激活的K+通道hlK1。由于经上皮Cl-分泌本质上是载体性的，因此离子转运体和通道正确定位于根尖膜或基底外膜是至关重要的。到目前为止，还没有关于hlK1组装和转运到质膜特别是基底外膜的机制的信息。我们的长期目标是确定负责hIK1正确组装，运输，基底侧定位和调控的分子基序。在Specific Aim A中，我们将定义c端亮氨酸拉链调节hlK1折叠、转运和调控的机制。此外，我们还证明，在n端亮氨酸拉链突变后，hIK1无法运输到质膜。在Specific Aim B中，我们将确定n端是否作为四聚域，n端是否与c端相互作用，以及突变是否影响通道调节。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，并确定这种共组装的生理和药理学后果。在Specific Aim C中，我们将定义分化上皮中hIK1基底外侧膜定位所需的分子基元。在这些研究中，我们将使用MDCK细胞系。我们将确定接头在hlK1定位中的作用，特别是上皮特异性mu1B。最后，我们将确定hIK1的内吞速率，并确定是否carbachol、胰岛素和EGF调节这一过程。我们所有的研究都将结合膜片钳、蛋白质生化和免疫荧光技术对异种表达hIK1的HEK293、LLC-PK1和MDCK细胞进行研究。总之，我们的研究将首次确定正确组装、运输和定位KCNN基因家族成员所需的分子基序。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，因此了解hIK1的运输和调节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