Regulation of hIK1 in Secretory Diarrhea

描述(申请人提供)：钙介导的激动剂是肠道分泌性腹泻的重要调节剂，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。事实上，据估计，美国每年的医疗费用为230亿美元。氯离子分泌过程中的一个关键步骤是激活基侧膜钙激活的钾通道hlK1。由于跨上皮氯离子的分泌本质上是矢量的，因此离子转运体和离子通道被正确地定位于根尖或基底膜是至关重要的。到目前为止，还没有关于hlK1被组装并运输到一般质膜，特别是基侧膜的机制的信息。我们的长期目标是确定负责正确组装、运输、碱侧定位和调节hIK1的分子基序。在特定的目标A中，我们将定义C末端亮氨酸拉链调节hlK1折叠和运输的机制。此外，我们还证明了在N端亮氨酸拉链突变后，hIK1不能运输到质膜上。在特定的目标B中，我们将确定N-末端是否作为四聚结构域，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构体，并确定这种共同组装的生理和药理学后果。在特定的目标C中，我们将定义hIK1在分化上皮中的基侧膜定位所需的分子基序。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，我们将定义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。我们的研究将结合膜片钳、蛋白质生化和免疫荧光技术对异源表达hIK1的HEK293、LLC-PK1和MDCK细胞进行。总之，我们的研究将首次确定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在钙离子介导的分泌性腹泻中起关键作用，并可能是CF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所帮助

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