FUNCTIONAL STUDIES OF THE NA,K-ATPASE

描述（来自申请人摘要的逐字）：我们的实验室是 对Na，K-ATP酶的各个方面感兴趣，包括结构-功能 关系和理解的异构体的调控表达， α和β亚基。我们特别感兴趣的是 这种酶发挥作用，最近开发出缺乏α 1的动物， α 2亚型。这些研究中特别重要的是， 每种亚型在心脏和骨骼肌中扮演着不同的角色 收缩。我们希望对这些研究采取后续行动，并继续进行其他研究 与这种酶在体内的作用有关。我们的具体目标 补助金将使用替代基因靶向方法来确定是否 强心苷结合位点的Na，K-ATP酶起着生物学作用。等 研究将有助于确定这种酶是否存在天然配体， 他们可能扮演的角色。在我们的第二个目标中，我们将利用这些动物 定义单个α亚型在介导阳性和 强心苷治疗的副作用。第三个目标涉及 了解每种α亚型的生理作用， 嵌合分析和组织特异性敲除。缺乏α 2的动物 同种型在出生后不能存活，这些动物提供了机会， 了解这种异构体所起的特殊生物学作用。为了 为了更全面地确定对致死表型有反应的组织，我们计划 进行嵌合研究，胚胎干细胞缺乏两个副本， α 2同种型并进行组织特异性敲除。这些方法 将有助于确定哪个器官系统（S）负责失败的 动物在出生后发育。这些研究也可能提供动物 与缺乏α 2亚型的组织结合， 在组织中的同种型。

DESCRIPTION (Verbatim from the Applicant's Abstract): Our laboratory is interested in various aspects of the Na,K-ATPase, including structure-function relationships and understanding the regulated expression of the isoforms of the alpha and beta subunits. We are especially interested in the physiological role this enzyme plays and have recently developed animals lacking the alpha1 and alpha2 isoforms. Of particular significance from these studies is the finding that each isoform plays a different role in heart and skeletal muscle contraction. We wish to follow up on these studies as well as pursue others that are related to the in vivo role of this enzyme. The specific aims of our grant are to use a replacement gene targeting approach to determine whether the cardiac glycoside binding site of the Na,K-ATPase plays a biological role. Such studies will help define whether natural ligands exist for this enzyme and the role that they might play. In our second aim, we will use these animals to define the role of individual alpha isoforms in mediating the positive and negative effects of cardiac glycoside therapy. A third aim relates to understanding the physiological role of each of the alpha isoforms using chimeric analysis and tissue-specific knockouts. Animals lacking the alpha2 isoform fail to survive past birth and these animals provide an opportunity for understanding the specific biological niche that this isoform plays. In order to more fully define the tissue responsile for the lethal phenotype, we plan to carry out chimeric studies with embryonic stem cells which lack both copies of the alpha2 isoform and to carry out tissue-specific knockouts. These approaches will help define which organ system(s) is responsible for the failure of the animals to develop past birth. These studies will also likely provide animals with tissues lacking the alpha2 isoform and make it possible to define the role of this isoform in that tissue.