Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

S.E. Faraj, W.M. Valsecchi, Mariela Ferreira-Gomes, Mercedes Centeno, Elina Malén Saint Saint Martin, Natalya Fedosova, Juan Pablo F.C. Rossi, Monica Montes, Rolando C. Rossi*

*Corresponding author for this work

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review


The Na +/K +-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na + for extracellular K + to the hydrolysis of ATP. The asymmetric distribution of Na + and K + is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers–Post model. It involves the presence of gates alternatively exposing Na +/K +-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K +, information is lacking about Na +-occluded intermediates, as occluded Na + was only detected in states incapable of performing a catalytic cycle, including two Na +-containing crystallographic structures. The current knowledge is that intracellular Na + must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na +-occluded intermediates, we isolated species with tightly bound Na + in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na + becomes spontaneously occluded in the E1 dephosphorylated form of the Na +/K +-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na + occlusion as it would have been expected; on the contrary, occluded Na + transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers–Post model for explaining the Na + transport pathway.

Original languageEnglish
Article number102811
JournalJournal of Biological Chemistry
Number of pages12
Publication statusPublished - Feb 2023


  • Albers–Post model
  • Na /K -ATPase
  • cation transport intermediates
  • enzyme kinetics
  • enzyme mechanism
  • epigallocatechin-3-gallate
  • membrane transport
  • sodium occlusion
  • sodium transport
  • Phosphorylation
  • Biocatalysis
  • Adenosine Triphosphate/metabolism
  • Cations, Monovalent/metabolism
  • Sodium/metabolism
  • Sodium-Potassium-Exchanging ATPase/chemistry
  • Potassium/metabolism
  • Animals
  • Cell Membrane/metabolism
  • Ion Transport
  • Kinetics


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