Abstract: Na+,K+-ATPases establish and maintain the vital electrochemical gradients for Na+ and K+ across animal cell membranes. The protein is a ternary complex composed of α, β and FXYD subunits, of which isoforms that fine-tune transport properties are expressed in a tissue-specific fashion. We report cryo-EM structures under active ATPase turn-over conditions of the ubiquitously expressed human α1β1FXYD1 and neuron-specific α3β1FXYD1 isoform complexes reconstituted in lipid nanodiscs and probe their specific functional and biophysical properties [1]. The data provides an extensive insight into Na+-transport of ATP-activated enzyme through distinct conformational states, including a long-sought sodium-bound and ADP-released phosphoenzyme intermediate, denoted [Na3]E2P, which has so far been elusive to structural studies. It represents the critical transition from inward to outward oriented states ([Na3]E1P-ADP to ([Na3]E2P) and immediately precedes the voltage-sensitive step of Na+ release.

We discuss models for the formation and destabilization of co-operative Na+ binding at the ion-binding sites and how it affects cytoplasmic ion gating leading to Na+ dependent phosphorylation, and subsequently the onset of extracellular Na+ release from the [Na3]E2P state.

 

The study also brings us to investigate and discuss the mechanism of differentiation in Na+ affinity of α3 compared to α1, which is physiologically important for how α3 becomes activated at elevated Na+ concentrations. Finally, we present the first structures of a disease-causing mutant form of α3, associated with Alternating Hemiplegia of Childhood (Q140L). The mutation compromises a specific phospholipid-binding pocket and impedes polyunsaturated phospholipid-mediated stimulation of Na+,K+-ATPase activity.