Na+,K+-ATPase with Disrupted Na+ Binding Sites I and III Binds Na+ with Increased Affinity at Site II and Undergoes Na+-Activated Phosphorylation with ATP

Nielsen, Hang N.; Holm, Rikke; Sweazey, Ryan; Andersen, Jens Peter; Artigas, Pablo; Vilsen, Bente

doi:10.3390/biom14010135

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Na⁺,K⁺-ATPase with Disrupted Na⁺ Binding Sites I and III Binds Na⁺ with Increased Affinity at Site II and Undergoes Na⁺-Activated Phosphorylation with ATP

by

Hang N. Nielsen

¹,

Rikke Holm

¹

,

Ryan Sweazey

²,

Jens Peter Andersen

¹,

Pablo Artigas

² and

Bente Vilsen

^1,*

¹

Department of Biomedicine, Aarhus University, DK-8000 Aarhus, Denmark

²

Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, TX 79430, USA

^*

Author to whom correspondence should be addressed.

Biomolecules 2024, 14(1), 135; https://doi.org/10.3390/biom14010135

Submission received: 12 December 2023 / Revised: 8 January 2024 / Accepted: 10 January 2024 / Published: 22 January 2024

(This article belongs to the Special Issue The Role of P-type ATPases in Health and Diseases)

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Abstract

Na⁺,K⁺-ATPase actively extrudes three cytoplasmic Na⁺ ions in exchange for two extracellular K⁺ ions for each ATP hydrolyzed. The atomic structure with bound Na⁺ identifies three Na⁺ sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na⁺ binds from the cytoplasmic side. It is usually assumed that the occupation of all three Na⁺ sites is obligatory for the activation of phosphoryl transfer from ATP. To obtain more insight into the individual roles of the ion-binding sites, we have analyzed a series of seven mutants with substitution of the critical ion-binding residue Ser777, which is a shared ligand between Na⁺ sites I and III. Surprisingly, mutants with large and bulky substituents expected to prevent or profoundly disturb Na⁺ access to sites I and III retain the ability to form a phosphoenzyme from ATP, even with increased apparent Na⁺ affinity. This indicates that Na⁺ binding solely at site II is sufficient to promote phosphorylation. These mutations appear to lock the membrane sector into an E₁-like configuration, allowing Na⁺ but not K⁺ to bind at site II, while the cytoplasmic sector undergoes conformational changes uncoupled from the membrane sector.

Keywords: Na⁺,K⁺-pump; Na⁺ site; K⁺ site; Na⁺ affinity; K⁺ affinity; P-type ATPase; mutagenesis; serine; phosphorylation; electrophysiology

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MDPI and ACS Style

Nielsen, H.N.; Holm, R.; Sweazey, R.; Andersen, J.P.; Artigas, P.; Vilsen, B. Na⁺,K⁺-ATPase with Disrupted Na⁺ Binding Sites I and III Binds Na⁺ with Increased Affinity at Site II and Undergoes Na⁺-Activated Phosphorylation with ATP. Biomolecules 2024, 14, 135. https://doi.org/10.3390/biom14010135

AMA Style

Nielsen HN, Holm R, Sweazey R, Andersen JP, Artigas P, Vilsen B. Na⁺,K⁺-ATPase with Disrupted Na⁺ Binding Sites I and III Binds Na⁺ with Increased Affinity at Site II and Undergoes Na⁺-Activated Phosphorylation with ATP. Biomolecules. 2024; 14(1):135. https://doi.org/10.3390/biom14010135

Chicago/Turabian Style

Nielsen, Hang N., Rikke Holm, Ryan Sweazey, Jens Peter Andersen, Pablo Artigas, and Bente Vilsen. 2024. "Na⁺,K⁺-ATPase with Disrupted Na⁺ Binding Sites I and III Binds Na⁺ with Increased Affinity at Site II and Undergoes Na⁺-Activated Phosphorylation with ATP" Biomolecules 14, no. 1: 135. https://doi.org/10.3390/biom14010135

APA Style

Nielsen, H. N., Holm, R., Sweazey, R., Andersen, J. P., Artigas, P., & Vilsen, B. (2024). Na⁺,K⁺-ATPase with Disrupted Na⁺ Binding Sites I and III Binds Na⁺ with Increased Affinity at Site II and Undergoes Na⁺-Activated Phosphorylation with ATP. Biomolecules, 14(1), 135. https://doi.org/10.3390/biom14010135

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Na⁺,K⁺-ATPase with Disrupted Na⁺ Binding Sites I and III Binds Na⁺ with Increased Affinity at Site II and Undergoes Na⁺-Activated Phosphorylation with ATP

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