REGULATION OF HIK1 IN SECRETORY DIARRHEA

钙介导的激动剂是肠道的重要调节剂 单独作用的分泌性腹泻(如副溶血性弧菌毒素)或 与cAMP介导的激动剂有协同作用。与以下项目相关的医疗成本 在美国，感染性腹泻每年估计有230亿美元。 各州。氯离子分泌过程中的一个关键步骤是激活 基底侧膜钙/2激活的钾通道(K/Ca)。因此，我们的龙 学期目标是了解这种K/Ca的生理调节 以及阐明K/Ca的药理调节剂在 疾病。在氯-分泌反应之间有明显的解离 和细胞内Ca/2+介导的Cl-分泌提示 除Ca/2外的第二信使是这一过程的重要调节器 K/Ca。我们证明了最近克隆的中间电导 K/Ca(HIK1)os在人结肠和呼吸道上皮细胞中的表达 该通道被依赖于PKA的磷酸化激活。第一 这项提案的主要目的是界定 HIK1的磷酸化调节其钙/2+依赖性调节。我们 将研究hiK1在非洲爪哇卵母细胞中的异源表达 双电极电压钳(TEVC)和切除膜片钳技术。 我们将把单一的PKA磷酸化共识位点突变为 证明这种丝氨酸的磷酸化在 调节依赖钙离子的hIK1的激活。我们还将研究 内源性hIK1在结肠和呼吸道上皮细胞中的表达，使用切除的补片- 钳制技术，以确定哪些磷脂酶在 去磷酸化HIK1。这些研究将允许一个完整的 了解细胞内cAMP升高是如何调节K/Ca的 与腹泻有关的氯-分泌反应涉及cAMP-和 依赖钙离子的激动剂。Clotrimazone是一种有效的hIK1受体阻滞剂 结肠上皮细胞，提示克霉唑可能作为一种 止泻药。此外，已知的是，Clotrimazone可以阻断Gardos通道 在红血球中。因为抑制Gardos通道会阻止RBC 镰状，克霉胺正在接受临床疗效评估 治疗镰状细胞性贫血。事实上，它看起来像是加尔多斯人 RBC的频道为hIK1。因此，这项提议的第二个主要目标是 在分子水平上定义克霉唑的作用机制 抑制hIKL。我们已经用嵌合体缩小了结合部位 在hIKl和相关SK通道之间构建。我们将建造 在使用点突变定义氨基之前的额外嵌合体 决定三氮杂环胺结合部位的酸。这些研究将 使用TEVC和切除的膜片钳技术。一个完整的 了解克霉唑的阻断机制将是很重要的。 用于治疗镰状细胞性贫血，可能还有腹泻。

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.