Regulation of the Na,K-ATPase by FXYD1

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CIRRI, Erica, 2012. Regulation of the Na,K-ATPase by FXYD1 [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Cirri2012Regul-19636, title={Regulation of the Na,K-ATPase by FXYD1}, year={2012}, author={Cirri, Erica}, address={Konstanz}, school={Universität Konstanz} }

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 extra¬cellular 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.<br />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 α1/His10-β1 and α2/His10-β1 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 α1/His10-β1 can be in vitro reconstituted with purified, detergent-solubilized human FXYD1 expressed in E. coli to obtain the α1/His10-β1/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.<br /><br />The biophysical techniques based on the fluorescence of external dyes available in our lab allow a thorough characterization of the transport cycle of the Na,K-ATPase. Among them, the electrochromic styryl dye RH421 enables us to monitor the ion movements inside the membrane domain of the enzyme, allowing the detection of ion binding and ion release during the transport cycle. Moreover, the time course of the signals provides information about the kinetics of the processes involved. In contrast, the voltage-sensitive dye Oxonol VI can be successfully applied to detect the ion transport of the Na,K-ATPase reconstituted in lipid vesicles.<br /><br />In a first step of the current study, the dye RH421 has been applied to the purified α1/His10-β1 and α2/His10-β1 preparations to ensure that it is suitable to investigate the ion-binding kinetics of detergent-solubilized ion pumps and that the functional properties of the purified recombinant enzymes do not differ significantly from those of the membrane-bound native Na,K-ATPase. Afterwards, the dye RH421 has been applied in steady-state and time-resolved kinetic measurements to characterize the effects of FXYD1 on the different partial reactions of the transport cycle of the α1/β1 isozyme of the Na,K-ATPase. These experiments have shown a single kinetic property affected by the presence of FXYD1: in both the enzyme conformations, E1 and P-E2, the Na+-binding affinity is increased of ~ 20-30%. In the final part of the study, the influence of the membrane and its lipid composition on the effect of FXYD1 on the Na+-binding affinity of the enzyme has been investigated with the voltage-sensitive dye Oxonol VI in proteoliposomes containing either α1/His10-β1 or α1/His10-β1/FXYD1. These experiments have revealed an unexpected role of the lipid environment surrounding the complex in the interaction of FXYD1 with the enzyme, probably related to the cytoplasmic segment of the regulatory protein. eng Cirri, Erica 2013-06-21T22:25:04Z Cirri, Erica Regulation of the Na,K-ATPase by FXYD1 2012 2012-06-27T11:33:09Z terms-of-use Regulation der Na,K-ATPase durch FXYD1

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