Regulation of the Na,K-ATPase by FXYD1

The Na,K-ATPase is an integral membrane protein present in virtually all animal cells, where it actively transports Na + and K + ions across the plasma membrane using ATP as energy source. For every ATP molecule hydrolyzed, the enzyme pumps three Na + ions out of and two K + ions into the cell. Because of its fundamental role in many physiological processes, the Na,K-ATPase is the target of specific regulatory mechanisms. Among them, the enzyme is modulated by the interaction with the so-called FXYD proteins, a group of short transmembrane polypeptides named after the invariant extracellular motif FXYD. All mammalian members of the FXYD family are known to associate with the Na,K-ATPase and modulate its properties in a tissue-and isozyme-specific way. FXYD1, also known as phospholemman, has been first identified as the major substrate for protein kinases A and C in the heart. Subsequently, it has been discovered to associate with specific isozymes of the Na,K-ATPase and modulate the enzyme activity in heart and skeletal muscle as well as kidneys and brain. So far, the effects of FXYD1 on the Na,K-ATPase have been investigated mainly in intact cells, both heterologous systems and native cells. These systems allow a better characterization of the physiological effects of FXYD1, but are of limited use for the investigation of the functional and structural interactions between FXYD1 and the enzyme. A purification procedure of the human α 1 /His 10-β 1 and α 2 /His 10-β 1 isozymes of the Na,K-ATPase expressed in yeast P. pastoris has been recently developed by the group of Steven Karlish at the Weizmann Institute of Science. The purified, detergent-solubilized α 1 /His 10-β 1 can be in vitro reconstituted with purified, detergent-solubilized human FXYD1 expressed in E. coli to obtain the α 1 /His 10-β 1 /FXYD1 complex. The purified recombinant preparations provide a system that enables us to work under well defined conditions and without interference by other cellular components. Unlike in native cells, the effects of FXYD1 on the different isozymes of the Na,K-ATPase can be investigated separately. Moreover, since the phosphorylation state of FXYD1 in the purified preparations is easily controllable, the functional role of the protein kinases-mediated phosphorylation of FXYD1 can be investigated. Therefore, these systems allow the performance of a detailed functional analysis of the effects of FXYD1 on the Na,K-ATPase. The biophysical techniques based on the fluorescence of external dyes available in …