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bioRxiv · 10.1101/2025.07.27.666930

Active Conformations of Neuronal Na+,K+-ATPase isoforms and a Disease-Causing Mutant

Christensen, M. E.·Habeck, M.·Katz, A.·Fruergaard, M. U.·Peleg, Y.·Pick, U.·Karlish, S. J. D.·Nissen, P.
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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 , {beta} 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 1{beta}1FXYD1 and neuron-specific 3{beta}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 long-sought sodium-bound phosphoenzyme intermediate, denoted [Na3]E2P. This hitherto elusive 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 investigated and discuss the mechanism of the physiologically important differentiation in Na+ affinity of 3 compared to 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 first structures of a disease-causing mutant form of 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.

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Christensen, M. E., Habeck, M., Katz, A., Fruergaard, M. U., Peleg, Y., Pick, U., Karlish, S. J. D., Nissen, P.. 2025-07-27. Active Conformations of Neuronal Na+,K+-ATPase isoforms and a Disease-Causing Mutant. https://doi.org/10.1101/2025.07.27.666930

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