Sodium pump regulation in health and disease

The sodium-potassium ATPase (NKA) is the ion motive ATP-dependent transporter that establishes Na+ and K+ gradients across the cell membrane, facilitating many physiological processes. In the heart, NKA activity is regulated by phospholemman (PLM), which PLM inhibits NKA by reducing its apparent affinity for Na+. In this study, we used time-correlated single-photon counting, FRET-microscopy, molecular dynamics simulations, and electrophysiology to investigate the stoichiometry of the native NKA-PLM regulatory complex. Our data suggest a stoichiometry of the NKA-PLM complex composed of two α subunits, two β subunits, and decorated with two PLM regulators. Protein-protein docking and molecular dynamics simulations generated a structural model of the regulatory complex. This model was characterized by contacts between cytoplasmic domains of α subunits, between β subunit extracellular domains, and between the highly-conserved α subunits’ M3 helix and the N-termini of opposing β subunit. The helix M3 shows significant movement during the pump's reaction cycle and mutations in M3 are associated with several diseases. Here, we studied the G301R mutant that is unable to correctly localize in the plasma membrane when expressed alone, but a milder G301A mutant localizes correctly. The G301R localization was improved in the presence of a WT pump suggesting a dominant negative effect of this mutation. Our simulations of the dimeric complexes revealed decreased affinity of the Na+ in position II, which is unbound during the simulated time window. Surprisingly, Na+ binding was not affected in the simulated WT or G301A/R monomers. Two-electrode voltage clamp experiments revealed decreased affinity of G301A for external Na+ and ion leak from G301R mutants supporting our simulation data. This combination of ex vivo and in silico experiments provides an insight into the quaternary structure of the NKA-PLM regulatory complex and helps to explain the mechanism of G301A/R mutation pathology.