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.