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. Here we report cryo-EM structures under active ATPase turn-over conditions of the ubiquitously expressed human α1β1FXYD1 and neuron-specific α3β1FXYD1 isoform complexes and probe their specific functional and biophysical properties. The data provides an extensive insight into Na+-transport of ATP-activated enzyme through four distinct conformational states, including a sodium-bound phosphoenzyme intermediate, denoted [Na3]E2P. This conformation reveals a crucial structural change that precedes Na+ release in the inward to outward (E1P-E2P) transition, within the general context of the sequential, active transport mechanism. We discuss the mechanism of the physiologically important differentiation in Na+ affinity of α3 compared to α1, the co-operative Na+ binding at the ion-binding sites, and the mechanistic aspects of cytoplasmic ion gating and extracellular Na+ release. Finally we present the 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.