FUNCTIONAL STUDIES OF THE NA,K-ATPASE

描述(摘自申请者摘要)：我们的实验室是 对Na，K-ATPase的各个方面感兴趣，包括结构和功能 关系和理解调节的表达的异构体 α亚基和β亚基。我们特别感兴趣的是生理作用 这种酶发挥作用，最近开发出缺乏α1和α1的动物 α2亚型。从这些研究中特别有意义的是发现 每种异构体在心脏和骨骼肌中扮演不同的角色 收缩。我们希望跟进这些研究，并继续进行其他研究。 这与这种酶在体内的作用有关。我们的具体目标是 格兰特将使用一种替代基因打靶的方法来确定 心脏糖苷结合部位的Na，K-ATPase起着生物学作用。是这样的 研究将有助于确定这种酶是否存在天然配体以及 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 明确个体阿尔法异构体在调节正性和 心脏糖苷类药物的不良反应。第三个目标与 了解每个阿尔法异构体的生理作用 嵌合分析和组织特异性基因敲除。缺乏字母的动物2 异构体在出生后不能存活，这些动物提供了一个机会 了解这种异构体所扮演的特定生物生态位。按顺序 为了更全面地确定致命性表型的组织反应，我们计划 对胚胎干细胞进行嵌合体研究，因为胚胎干细胞既缺乏 α2亚型并进行组织特异性基因敲除。这些方法 将有助于确定哪个器官系统(S)应为 动物在过去的出生中发育。这些研究还可能提供动物 缺乏α2异构体的组织，从而有可能确定其作用 在那个组织中的这种异构体。

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.