REGULATION OF HIK1 IN SECRETORY DIARRHEA

钙介导的激动剂是肠促胰岛素释放的重要调节剂。 单独作用的分泌性腹泻（例如，副溶血性弧菌毒素）或 与cAMP介导的激动剂协同作用。与下列疾病有关的医疗费用 据估计，美国每年因感染性腹泻造成的损失为230亿美元， States. Cl-分泌过程中的关键步骤是激活 基底外侧膜Ca/2激活的K+通道（K/Ca）。因此，我们的长期 长期目标是了解这种K/Ca的生理调节 以及阐明K/Ca药理学调节剂的作用， 疾病Cl-分泌反应与 和细胞内Ca/2+在Ca/2+介导的Cl-分泌过程中的作用，表明 Ca/2以外的第二信使是这一过程的重要调节剂， 钾/钙我们证明，最近克隆的中间电导 K/Ca（hIKl）os在人结肠和气道上皮中表达， 该通道被PKA依赖性磷酸化激活。第一 这项建议的主要目的是确定机制， hIK 1的磷酸化调节其Ca/2+依赖性调节。我们 将研究hiKl在爪蟾卵母细胞中异源表达， 双电极电压钳（TEVC）和离体膜片钳技术。 我们将突变单个PKA磷酸化共有位点， 证明了这种丝氨酸的磷酸化在 调节hIKl的Ca/2+依赖性活化。我们还将研究 结肠和气道上皮中的内源性hIKl，使用切除的贴片- 钳技术，以确定哪些磷酸酶是重要的， 使hIKl去磷酸化。这些研究将使一个完整的 了解增加细胞cAMP如何调节K/Ca， 与涉及cAMP-和 Ca/2+依赖性激动剂。克霉宗是一种有效的hIKl阻断剂， 结肠上皮细胞，表明克霉唑可能是有用的， 抗疟药此外，已知氯曲马宗会阻断Gardos通道 在红细胞中。由于Gardos通道的抑制阻止了RBC 镰状化，正在评估氯曲马宗在 治疗镰状细胞性贫血事实上，它似乎是加尔多人 RBC的通道是hIKl。因此，这项建议的第二个主要目的是 在分子水平上定义克霉宗 抑制hIKl。我们已经缩小了结合位点使用嵌合 hIKl和相关SK通道之间的构建体。了建设一 在使用点突变来定义氨基酸之前， 确定氯曲马松结合位点的酸。这些研究将 使用TEVC和切除的膜片钳技术进行。一个完整 了解氯曲马松阻滞的机制将是重要的 用于治疗镰状细胞性贫血和潜在的腹泻。

Calcium-mediated agonists are important modulators of intestinal secretory diarrhea acting alone (e.g., Vibrio parahaemolyticus toxin) or in synergism with cAMP-mediated agonists. Medical costs associated with infectious diarrhea 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 Ca/2-activated K+ channel (K/Ca). Thus, our long term goals are to understand the physiological regulation of this K/Ca as well as to clarify the role of pharmacological modulators of K/Ca in disease. There is a clear dissociation between the Cl-secretory response and intracellular Ca/2+ during Ca/2+-mediated Cl- secretion suggesting that second messengers other than Ca/2 are important modulators of this K/Ca. We demonstrate that the recently cloned intermediate conductance K/Ca (hIKl) os expressed in both human colonic and airway epithelial and this channel is activated by PKA-dependent phosphorylation. The first major aim of this proposal is to define the mechanism whereby phosphorylation of hIKl modulates its Ca/2+- dependent regulation. We will study hiKl heterologously expressed in Xenopus oocytes using both two-electrode voltage-clamp (TEVC) and excised patch-clamp techniques. We will mutate the single PKA phosphorylation consensus site to demonstrate that phosphorylation of this serine is critical in modulating the Ca/2+-dependent activation of hIKl. We will also study endogenous hIKl in colonic and airway epithelia, using excised patch- clamp techniques, to determine which phospatases are important in dephosphorylating hIKl. These studies will allow for a complete understanding of how increasing cellular cAMP modulates K/Ca and hence the Cl-secretory response associated with diarrhea involving cAMP- and Ca/2+-dependent agonists. Clotrimazone is a potent blocker of hIKl in colonic epithelia, suggesting that clotrimazole may be useful as an antidiarrheal. Also, clotrimazone is known to block the Gardos channel in red blood cells. As inhibition of the Gardos channel prevents RBC sickling, clotrimazone is being evaluated for clinical efficacy in the treatment of sickle cell anemia. Indeed, it appears as the Gardos channel of RBCs is hIKl. Thus, the second major aim of this proposal is to define, at a molecular level, the mechanism by which clotrimazone inhibits hIKl. We have narrowed the binding site down using chimeric constructs between hIKl and the related SK channels. We will construct additional chimeras before using point mutations to define the amino acids which determine the clotrimazone binding site. These studies will be carried out using TEVC and excised patch-clamp techniques. A complete understanding of the mechanism of clotrimazone block will be important for the treatment of sickle cell anemia and, potentially, diarrhea.