MOLECULAR AND PHYSIOLOGIC STUDIES OF A COLONIC K ATPASE

描述：本申请提出继续生理和 大鼠远端结肠钾吸收的分子研究。 的PI 在大鼠远端结肠中发现了一个活跃的钾吸收过程 与顶端K/H交换有关，由一种新的H，K-ATP酶提供能量。 的 PI还表明，这一过程被醛固酮和 膳食钾耗竭 PI最近的研究集中在 H，K-ATP酶活性随着钾吸收的增加而增加， 分子和生理水平的研究。 PI报告称， 大鼠远端结肠H，K-ATP酶对哇巴因部分敏感，提示 可能存在两种不同的钾吸收过程， H，K-ATP酶亚型。 他的小组最近成功地克隆了 HCKalpha1 cDNA。 该cDNA在SF 9细胞中的表达表明哇巴因 与其他使用卵母细胞的研究相比， 表达系统 因此，在这项建议中，他计划确定 转染后的转运蛋白cDNA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性β亚基是否是难以捉摸的 和推定的β亚基用于最佳功能表达。 主要研究者有 确定了一个哇巴因敏感的K依赖性pHi恢复过程中， 结肠隐窝 PI假设该pHi调节将 通过膳食K消耗进行修改，因此将代表第二个 H，K-ATP酶功能。 为了鉴定第二个结肠H，K-ATP酶，PI将 使用克隆策略，基于其可能通过饮食上调 钾耗尽，其身份的H-3结构域区域，而不是 HCKalpha 1 cDNA 5 ′端及其在结肠中的特异性定位 地穴 也有人假设，饮食中钾的缺乏可能 调节第二结肠H，K-ATP酶（HCKa 2和/或HCKab） 膜和/或电中性KCl共转运在基底外侧膜。 PI计划利用细胞和分子生物学研究来完成他的研究。 四个具体目标，旨在获得更多的了解结肠钾 吸收 这项建议当然与临床医学有关， 将增强我们对钾转运的了解

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.