MOLECULAR BASIS OF CARDIAC GLYCOSIDE AFFINITY

Na，K泵（即，Na，K-ATPase）被认为是主要的 强心苷如洋地黄的药理学受体 和哇巴因。 它是一种跨膜蛋白复合物， 介导Na+和K+的跨膜交换， 代谢能量的消耗。 关键职能 泵的产生和维护 Na+和K+在等离子体中的电化学梯度 膜，Ca++，糖和氨基酸的吸收，通过 共同运输系统，以及盐和水的运输 上皮细胞 强心苷可抑制钠钾泵， 在给定的物种中，哇巴因亲和力存在差异， 组织间的差异可能高达500倍。 的基础 这种可变性还没有得到很好的理解。 还有组织 不同亚型表达的变异性 钠钾泵的组成亚基。 该项目将测试 假设哇巴因亲和力变化的基础是 不同亚型的α和 β亚单位 哇巴因的亚基组成和表达 将在许多培养的细胞系中检测亲和力， RNA杂交分析和酶测定，以关联 存在具有给定结合亲和力的特异性同种型。 DNA- 介导的基因转移将用于引入各种 同种型进入受体细胞系，以直接观察它们的作用 哇巴因的亲和力 表达的mRNA编码的是 天然亚基将通过杂交分析监测， 转移外源同种型，以确定其对 天然同种型的调节。 根据这些研究， 哇巴因亲和力的组织变异性的分子基础将是 阐明。

The Na,K-pump (ie., Na,K-ATPase) is thought to be the major pharmacological receptor for cardiac glycosides such as digitalis and ouabain. It is a membrane-spanning protein complex that mediates the transmembrane exchange of Na+ and K+ at the expense of metabolic energy. Critical functions served by the pump include the generation and maintenance of the electrochemical gradients of Na+ and K+ across the plasma membrane, the absorption of Ca++, sugars and amino acids via cotransport systems, and the transport of salts and water across epithelia. The Na,K-pump is inhibited by cardiac glycosides, and within a given species, there is variation in ouabain affinity between tissues that may be as great as 500-fold. The basis for this variability is not well understood. There is also tissue variability in the expression of different isoforms of the component subunits of the Na,K-pump. This project will test the hypothesis that the basis of the variation in ouabain affinity is the cell-specific expression of different isoforms of the alpha and beta subunits. The subunit composition and the expressed ouabain affinity will be examined in a number of cultured cell lines by RNA hybridization analysis and enzymatic assays to correlate the presence of specific isoforms with a given binding affinity. DNA- mediated gene transfer will be used to introduce the various isoforms into recipient cell lines to observe directly their effects on ouabain affinity. Expression of the mRNAs that encode the native subunits will be monitored by hybridization analysis after transfer of a foreign isoform to determine its effects on the regulation of the native isoforms. From these studies, the molecular basis of the tissue variability in ouabain affinity will be elucidated.