MOLECULAR AND PHYSIOLOGIC STUDIES OF A COLONIC K+ ATPASE

这一申请建议继续研究钾的运输 这是1975年首次发起的。本提案计划 K-ATP酶的分子和生理特性， 大鼠远端结肠。在这一过程中， 并通过醛固酮和膳食钾来增强 耗尽PI提出其细胞机制是K-H 由一种新型的K-ATP酶激发的交换。PI最近克隆了一个cDNA 其推导的氨基酸序列似乎代表了一种新的 a亚基P型ATP酶。该提案计划研究表达这种cDNA 在用合适的结肠细胞系进行的转染研究中， 该cDNA是否编码假定的K-ATP酶;如P型ATP酶 通常需要α和β亚基来完全表达， 有必要开始研究克隆这种β亚基， 推定的K-ATPase。针对推导的氨基酸的合成片段的抗体 该cDNA的酸性序列将允许鉴定K-ATP酶 蛋白免疫化学和检查其细胞分布。我们计划 继续进行原位杂交研究， 传播其信息。我们还计划确定监管 醛固酮和/或钾对K-ATP酶增强作用的控制 通过检查它们对信息、蛋白质和酶的影响， 活动 这些实验还将建立细胞机制， 钾离子在顶侧和基底侧细胞膜上的移动。在这些 建议的研究，我们必须确定适当的细胞系， 表达和调控研究;我们计划确定钾 主要通过顶端和基底侧膜囊泡运输。

This application proposes continuation of studies of potassium transport that were first initiated in 1975. The present proposal plans the molecular and physiologic characterization of the K-ATPase present in the rat distal colon. Active potassium absorption has been identified in this epithelium and is enhanced by both aldosterone and dietary potassium depletion. The PI has proposed that its cellular mechanism is a K-H exchange energized by a novel K-ATPase. The PI has recently cloned a cDNA from distal whose deduced amino acid sequence appears to represent a novel a subunit P-type ATPase. This proposal plans studies to express this cDNA in transfection studies with a suitable colon cell line to determine whether this cDNA encodes the putative K-ATPase; as P-type ATPases frequently require both alpha and beta subunits for full expression it may be necessary to initiate studies to clone the beta subunit of this putative K-ATPase. Antibodies to synthetic segments of the deduced amino acid sequence of this cDNA will permit identification of the K-ATPase protein immunochemically and to examine its cellular distribution. We plan to continue in situ hybridization studies to establish the cellular distribution of its message. We also plan to determine the regulatory control of the enhancement of K-ATPase by aldosterone and/or potassium deprivation by examining their effects on its message, protein, and enzyme activity. These experiments will also establish the cellular mechanism of potassium movement across apical and basolateral cell membranes. In these proposed studies we must identify appropriate cell lines for the expression and regulation studies; and we plan to determine potassium transport primarily by both apical and basolateral membrane vesicles.