The NKA Interactome in Health and Disease

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Project Summary/Abstract: The Na/K-ATPase (NKA), or “sodium pump”, is an enzyme that transports ions across cell membranes. Between 30-70% of the cell’s ATP budget is spent by the NKA to create strong chemical gradients for sodium and potassium. These gradients control cell volume, support electrical signaling (action potentials) and energize many other transport processes. Disruption of sodium handling is associated with diseases including cardiac hypertrophy/failure, Parkinson’s disease, schizophrenia, and Alzheimer’s disease. Various cell types in diverse tissues of the body have very different requirements for Na/K transport, and transport rates must respond to physiological challenges (e.g. exercise). To adapt NKA to these different conditions, the pump is regulated by tissue-specific transmembrane peptides that increase or decrease the pump’s enzymatic activity. In Aim 1 of the project we will focus on the normal physiological regulation of NKA by FXYDs, testing how FXYD proteins bind and stabilize specific NKA conformations to alter NKA transport kinetics. We will use novel spectroscopic assays to investigate the structure and stability of different NKA-FXYD regulatory complexes. We will investigate how physical coupling of two pumps into a single functional unit can allow faster overall cycling. Aim 2 is the translational arm of the research project. The research team will investigate how NKA regulation by FXYDs becomes disordered in disease, using proteomic analysis to compare the NKA-FXYD interactome in healthy and failing human heart tissue. We will determine whether fragments of digested membrane proteins may disrupt NKA function in disease. We will also test the feasibility of using gene delivery of an inhibitory species (FXYD1) as a therapy to improve the contraction strength of a failing heart. Finally, we will compare FXYDs that activate NKA and FXYDs that inhibit NKA. The project brings together 6 different investigators that specialize in different complementary approaches. The laboratory of the project PI, Prof. Seth Robia, uses fluorescence spectroscopy and cell physiology to investigate the structure, affinity, and function of NKA-FXYD complexes in cardiac myocytes and heterologous cells. He works closely with two Loyola University Chicago colleagues: Jonathan Kirk (Co-I) and Pete Kekenes-Huskey (Co-I). Prof. Kirk will use proteomics approaches such as mass spectrometry to discover post- translational modifications and novel NKA regulators. Prof. Kekenes-Huskey will assist with computational modeling of the protein complexes. Prof. Julie Bossuyt (MPI) will investigate a-a coupling, conducting microscopy and biochemistry experiments to identify the interaction interface. Prof. Artigas (Co-I) at Texas Tech will perform detailed electrophysiological analyses of FXYD proteins and membrane protein fragments. Prof. Razvan Cornea (Consultant) is an expert in membrane protein biophysics. He will advise the team on all aspects of the project. Together, the collaborators will uncover important new information about NKA-FXYD structure/function relationships, and learn how these mechanisms become disordered in disease.