<p>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 <b>α</b>, <b>β</b> 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 <b>α</b>1<b>β</b>1FXYD1 and neuron-specific <b>α</b>3<b>β</b>1FXYD1 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 <b>α</b>3 compared to <b>α</b>1, 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 <b>α</b>3, 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.</p>