MOLECULAR BASIS OF CARDIAC GLYCOSIDE AFFINITY

Na，K-泵(即Na，K-ATPase)被认为是主要的 洋地黄等强心苷的药理受体 还有哇巴因。它是一种跨膜的蛋白质复合体， 介导Na+和K+的跨膜交换 消耗新陈代谢能量。服务的关键职能 泵的产生和维护包括 Na~+和K~+在等离子体中的电化学梯度 膜，钙、糖和氨基酸的吸收通过 联运系统，以及盐分和水的跨境运输 上皮细胞。Na，K泵被心脏糖苷类药物抑制，并且 在一个给定的物种内，哇巴因亲和力存在差异 组织之间的距离可能高达500倍。的基础 这种可变性还没有被很好地理解。也有纸巾 不同亚型基因表达的可变性 Na，K-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 α和α不同异构体的细胞特异性表达 β亚基。哇巴因亚基组成及表达产物 许多培养细胞系的亲和力将通过以下方式进行检测 RNA杂交分析和酶分析相关联 具有给定结合亲和力的特定异构体的存在。DNA- 介导性基因转移将被用来引入各种不同的 异构体进入受体细胞系，直接观察它们的效果 关于哇巴因亲和力。编码基因的mRNAs的表达 天然亚基将通过杂交分析进行监测 外源异构体的转移，以确定其对 天然异构体的调节。从这些研究中， 哇巴因亲和力的组织变异性的分子基础将是 已澄清。

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.