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

doi:10.1016/j.jbc.2022.102811

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 af dette arbejde

Institut for Biomedicin - Forskning og uddannelse, Vest

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

4 Citationer (Scopus)

Abstract

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.

Originalsprog	Engelsk
Artikelnummer	102811
Tidsskrift	Journal of Biological Chemistry
Vol/bind	299
Nummer	2
Antal sider	12
ISSN	0021-9258
DOI	https://doi.org/10.1016/j.jbc.2022.102811
Status	Udgivet - feb. 2023

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10.1016/j.jbc.2022.102811Licens: CC BY

Citationsformater

Faraj, S. E., Valsecchi, W. M., Ferreira-Gomes, M., Centeno, M., Saint Martin, E. M. S., Fedosova, N., Rossi, J. P. F. C., Montes, M., & Rossi, R. C. (2023). Measurements of Na⁺-occluded intermediates during the catalytic cycle of the Na⁺/K⁺-ATPase provide novel insights into the mechanism of Na⁺ transport. Journal of Biological Chemistry, 299(2), Artikel 102811. https://doi.org/10.1016/j.jbc.2022.102811

@article{26a46206ccb347549b877b1ba87671b0,

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

abstract = "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.",

keywords = "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",

author = "S.E. Faraj and W.M. Valsecchi and Mariela Ferreira-Gomes and Mercedes Centeno and {Saint Martin}, {Elina Mal{\'e}n Saint} and Natalya Fedosova and Rossi, {Juan Pablo F.C.} and Monica Montes and Rossi, {Rolando C.}",

year = "2023",

month = feb,

doi = "10.1016/j.jbc.2022.102811",

language = "English",

volume = "299",

journal = "Journal of Biological Chemistry",

issn = "0021-9258",

publisher = "American Society for Biochemistry and Molecular Biology Inc.",

number = "2",

}

Faraj, SE, Valsecchi, WM, Ferreira-Gomes, M, Centeno, M, Saint Martin, EMS, Fedosova, N, Rossi, JPFC, Montes, M & Rossi, RC 2023, 'Measurements of Na⁺-occluded intermediates during the catalytic cycle of the Na⁺/K⁺-ATPase provide novel insights into the mechanism of Na⁺ transport', Journal of Biological Chemistry, bind 299, nr. 2, 102811. https://doi.org/10.1016/j.jbc.2022.102811

Measurements of Na⁺-occluded intermediates during the catalytic cycle of the Na⁺/K⁺-ATPase provide novel insights into the mechanism of Na⁺ transport. / Faraj, S.E.; Valsecchi, W.M.; Ferreira-Gomes, Mariela et al.
I: Journal of Biological Chemistry, Bind 299, Nr. 2, 102811, 02.2023.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review

TY - JOUR

T1 - Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

AU - Faraj, S.E.

AU - Valsecchi, W.M.

AU - Ferreira-Gomes, Mariela

AU - Centeno, Mercedes

AU - Saint Martin, Elina Malén Saint

AU - Fedosova, Natalya

AU - Rossi, Juan Pablo F.C.

AU - Montes, Monica

AU - Rossi, Rolando C.

PY - 2023/2

Y1 - 2023/2

N2 - 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.

AB - 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.

KW - Albers–Post model

KW - Na /K -ATPase

KW - cation transport intermediates

KW - enzyme kinetics

KW - enzyme mechanism

KW - epigallocatechin-3-gallate

KW - membrane transport

KW - sodium occlusion

KW - sodium transport

KW - Phosphorylation

KW - Biocatalysis

KW - Adenosine Triphosphate/metabolism

KW - Cations, Monovalent/metabolism

KW - Sodium/metabolism

KW - Sodium-Potassium-Exchanging ATPase/chemistry

KW - Potassium/metabolism

KW - Animals

KW - Cell Membrane/metabolism

KW - Ion Transport

KW - Kinetics

U2 - 10.1016/j.jbc.2022.102811

DO - 10.1016/j.jbc.2022.102811

M3 - Journal article

C2 - 36539036

SN - 0021-9258

VL - 299

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

IS - 2

M1 - 102811

ER -

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

Abstract

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Citationsformater

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