MOLECULAR AND PHYSIOLOGIC STUDIES OF A COLONIC K ATPASE

描述：此应用程序建议继续生理学和 大鼠远端结肠钾吸收的分子研究。《少年派》 在大鼠远端结肠中发现了一种活性钾吸收过程 与一种新的H，K-ATPase激活的顶端K/H交换有关。这个 PI还表明，这一过程被醛固酮和 饮食中钾的耗竭。PI最近的研究重点是将 H，K-ATPase活性与钾吸收的关系 分子和生理水平的研究。私家侦探报告说 大鼠远端结肠H，K-ATPase对哇巴因部分敏感，指向 可能存在两种不同的钾吸收过程和 H，K-ATPase亚型。他的团队最近成功地克隆了 HCKalpha1基因。该基因在SF9细胞中的表达显示哇巴因 与其他组使用卵母细胞的研究相比，不敏感 表达系统。因此，在这项提案中，他计划确定 转运蛋白cDNAs转导后的生理功能 哺乳动物细胞表达系统。还建议进行研究，以确定 最近克隆的大鼠结肠特异的β亚基是否难以捉摸 并推测为最佳功能表达的β亚基。少年派有 发现了哇巴因敏感的钾依赖的phi恢复过程 结肠隐窝。PI假设这一PHI规则将是 由于饮食中钾的消耗而改变，因此将代表第二个 H，K-ATPase功能。为了鉴定第二个结肠H，K-ATPase，PI将 使用基于饮食可能上调的克隆策略 钾的耗竭，它的同一性是H-3结构域，而不是 HCKalpha1基因的5‘末端及其在结肠中的独占定位 地窖。它还假设，饮食中的钾缺乏可能 调节结肠顶端第二个H，K-ATPase(HCKa2和/或HCKab) 膜和/或电中性钾在基侧膜上的共转运。 PI计划利用细胞和分子生物学研究来完成他的 旨在加深对结肠钾的了解的四个具体目标 吸收。这项建议当然与临床医学和 将增强我们对钾运输的了解。

DESCRIPTION: This application proposes continuation of physiologic and molecular studies of potassium absorption in the rat distal colon. The PI has identified an active K absorptive process in the rat distal colon associated with an apical K/H exchange energized by a novel H,K-ATPase. The PI has also shown that this process is upregulated by both aldosterone and dietary K depletion. The PI's recent studies have focused on correlating the H,K-ATPase activity with the upregulation of K absorption both at the molecular and physiologic levels of study. The PI has reported that H,K-ATPase of rat distal colon is partially ouabain sensitive, pointing to the possible presence of two different potassium absorptive processes and H,K-ATPase isoforms. His group was recently successful in cloning the HCKalpha1 cDNA. The expression of this cDNA in SF9 cells showed ouabain insensitivity in contrast to studies from other groups using the oocyte expression system. In this proposal he, therefore, plans to determine the physiological function of the transporter cDNAs after transfection in a mammalian cell expression system. Studies are also proposed to determine whether the recently cloned rat colon-specific beta subunit is the elusive and putative beta subunit for optimal functional expression. The PI has identified an ouabain-sensitive K-dependent pHi recovery process in the colonic crypt. The PI hypothesizes that this pHi regulation will be modified by dietary K depletion and will, therefore, represent the second H,K-ATPase function. To identify the second colonic H,K-ATPase, the PI will use a cloning strategy based on its probable upregulation by dietary potassium depletion, its identity to the H-3 domain region rather than to the 5' end of HCKalpha1 cDNA and its exclusive localization in colonic crypts. It is also hypothesized that dietary potassium depletion might regulate the second colonic H,K-ATPase (HCKa2 and/or HCKab) at the apical membrane and/or electroneutral KCl cotransport at the basolateral membrane. The PI plans to employ cell and molecular biology studies to accomplish his four specific aims aimed at gaining more understanding of colonic potassium absorption. This proposal certainly has relevance to clinical medicine and would enhance our knowledge of potassium transport.