Functional Studies of the Na,K-ATPase

描述（由申请人提供）：本申请建议继续我们对Na， k - atp酶的研究，并针对几个正在进行的目标。其中之一是检测该酶在物种间高度保守的alpha2或alpha3亚型的心脏糖苷结合位点是否发挥生物学作用。这些研究将为许多实验室观察到的内源性心脏糖苷（如内源性瓦巴因）是否具有生理意义提供见解。另一个目的是通过分析组织特异性alpha2异构体敲除动物来确定alpha2异构体在心脏和血管平滑肌中的作用。我们资助的具体目的是：(1)确定Na， k - atp酶的心脏糖苷结合位点是否具有生物学意义以及内源性心脏糖苷是否发挥生理作用。为了实现这一目标，将在已知会增加内源性心脏糖苷水平的条件下，如acth诱导的高血压，对表达抗瓦阿巴因α 2或α 3亚型的基因工程小鼠进行分析。如果抗瓦阿因小鼠的反应与野生型动物不同，这将提示内源性瓦阿因的生理作用。然而，如果具有瓦巴因抗性的alpha2或alpha3亚型小鼠的反应与野生型动物相似，这将削弱内源性心脏糖苷具有生理意义的假设。(2)利用组织特异性敲除确定alpha2亚型的具体作用。我们已经培育出了alpha2同种异构体基因两侧有loxP位点的小鼠，并将这些小鼠与在心脏特异性启动子控制下携带cre -重组酶的动物进行了配对。现在，我们将在平滑肌启动子的控制下，将alpha2异构体loxP动物与表达cre -重组酶的动物交配。这些配对将为那些只在心脏或平滑肌中缺少α 2亚型的动物提供帮助，这将使我们能够研究这种亚型在心血管功能中的具体作用。

DESCRIPTION (provided by applicant): This application proposes to continue our studies of the Na,K-ATPase and is directed toward several ongoing goals. One of these is to test whether the cardiac glycoside binding site of the alpha2 or alpha3 isoforms of this enzyme, which is highly conserved among species, plays a biological role. Such studies will provide insight as to whether endogenous cardiac glycosides such as endogenous ouabain observed by many laboratories is physiologically significant. Another objective is to define the role of the alpha2 isoform in heart and vascular smooth muscle by analyzing tissue-specific alpha2 isoform knockout animals. The specific aims of our grant are (1) To determine whether the cardiac glycoside binding site of the Na,K-ATPase has biological significance and whether endogenous cardiac glycosides play a physiological role. To accomplish this, genetically engineered mice expressing either ouabain-resistant alpha2 or alpha3 isoforms will be analyzed under conditions known to increase the levels of endogenous cardiac glycosides such as ACTH-induced hypertension. If the ouabain-resistant mice respond differently from wild type animals, this will suggest a physiological role for endogenous ouabain. However, if mice with ouabain resistant alpha2 or alpha3 isoforms respond similarly to wild type animals, this will detract from the hypothesis that endogenous cardiac glycosides have physiological significance. (2) To define the specific role of the alpha2 isoform using tissuespecific knockouts. We have already developed mice where the alpha2 isoform gene is flanked by loxP sites and we have mated these to animals carrying Cre-recombinase under the control of a cardiac-specific promoter. We will now mate the alpha2 isoform loxP animals with those expressing Cre-recombinase under the control of a smooth muscle promoter. These matings will provide animals where the alpha2 isoform is missing only in heart or smooth muscle and this will allow us to study the specific role of this isoform in cardiovascular function.