H+/K+ ATPASE AND ITS GENES

H /K ATP酶及其基因

• 批准号: 2140991
• 负责人: GARY EDWARD SHULL
• 金额: $ 16.74万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-03-01 至 1997-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140991
• 关键词: DNA footprinting; acid base balance; aldosterone; electrolyte balance; embryonic stem cell; enzyme mechanism; enzyme structure; gastric juice; gastrointestinal absorption /transport; gel mobility shift assay; genetic regulation; genetically modified animals; hydrogen; immunocytochemistry; isozymes; laboratory mouse; laboratory rabbit; laboratory rat; membrane transport proteins; molecular cloning; northern blottings; nucleic acid sequence; polymerase chain reaction; potassium; protein structure function; site directed mutagenesis; sodium potassium exchanging ATPase; structural genes

Our long-term objectives are to determine the biological roles, structure-function relationships, and genetic regulatory mechanisms for members of the H,K-ATPase family of enzymes. H,K-ATPases are ATP-dependent pumps that mediate the secretion of acid in stomach, absorption of potassium in colon, and both acid secretion and potassium absorption in kidney. Thus, they play essential roles in the digestion of food and in the maintenance of both acid-base balance and potassium homeostasis. A better understanding of the biological functions of these enzymes, the genetic and physiological mechanisms controlling their expression, and their structure-function relationships should lead to improved treatment for a variety of gastrointestinal and kidney diseases. We recently isolated and characterized cDNAs encoding the distal colon H,K-ATPase alpha-subunit and have obtained evidence demonstrating that it is expressed in kidney and suggesting that it may correspond to the H,K-ATPase of the distal nephron. The gene for the distal colon H,K-ATPase alpha-subunit will be analyzed in order to obtain basic information needed for gene regulation studies, and a cDNA encoding its beta-subunit will be isolated and characterized. PCR and genomic cloning studies will be performed to identify the rat homolog of the human alpha-D gene, which seems to encode a third distinct H,KATPase isoform. Changes in distal colon H,K-ATPase mRNA and protein levels in response to alterations in acid-base balance, potassium depletion and hormone treatment will be analyzed by Northern blot and immunological techniques. The cell-type specificity and membrane location of the enzyme will be determined by in situ hybridization and immunocytochemistry. Embryonic stem cell technology will be used to prepare a mouse in which the distal colon H,K-ATPase gene has been disrupted, thereby allowing an evaluation of the physiological effects of the absence of this gene, which would be expected to include disturbances in acid-base balance and potassium homeostasis. The regulatory elements of the genes encoding the alpha and beta subunits of the gastric H,K-ATPase and the alpha subunit of the distal colon H,K-ATPase will be identified using transgenic mice, DNA footprint analysis and mobility shift assays. Structure-function relationships of both the gastric and distal colon H,K-ATPases, with particular emphasis on the domains involved in ion-specificity and binding of drugs such as omeprazole and SCH28080 will be examined using H,K-ATPase/Na,K-ATPase chimeras and site-directed mutagenesis techniques.

我们的长期目标是确定生物学作用， 结构功能关系和遗传调节机制 H，K-ATPase酶家族的成员。 h，k-atpases是 介导胃中酸分泌的ATP依赖性泵， 钾在结肠中的吸收，以及酸分泌和钾 肾脏吸收。 因此，他们在消化中起着重要的作用 食物和酸碱平衡和钾的维持 稳态。 更好地了解这些生物学功能 酶，控制其的遗传和生理机制 表达及其结构功能关系应导致 改善了各种胃肠道和肾脏疾病的治疗方法。 我们最近分离和表征编码远端结肠的cDNA h，k-atpase alpha-subunit，并获得了证据证明它 用肾脏表达，表明它可能与 h，远端肾单位的k-atpase。 远端结肠的基因 H，K-ATPase Alpha-Subunit将进行分析，以获得基本 基因调节研究所需的信息，以及编码其的cDNA Beta-Subunit将被隔离和表征。 PCR和基因组克隆 将进行研究以识别人类的大鼠同源物 α-D基因似乎编码第三个不同的H，Katpase同工型。 远端结肠H，K-ATPase mRNA和蛋白质水平的变化响应于 酸碱平衡，钾耗竭和激素的改变 治疗将通过北印迹和免疫学技术进行分析。 酶的细胞类型特异性和膜位置将是 通过原位杂交和免疫细胞化学确定。 胚胎 干细胞技术将用于制备远端的鼠标 结肠H，K-ATPase基因已被破坏，从而进行评估 缺乏该基因的生理影响，这将是 预计将包括酸碱平衡和钾的干扰 稳态。 编码α和 胃H，K-ATPase和Alpha亚基的β亚基 将使用转基因小鼠DNA鉴定远端结肠H，K-ATPase 足迹分析和移动性转移分析。 结构功能 胃结肠和远端H，k-atpases的关系，与 特别强调与离子特异性有关的领域和 将使用Omeprazole和Sch28080等药物的结合使用 H，K-ATPase/Na，K-ATPase Chimeras和位置定向的诱变技术。