The Occlusion of Rb+ in the Na+/K+-ATPase

González-Lebrero, Rodolfo M.; Kaufman, Sergio B.; Montes, María Luciana; Nørby, Jens G.; Garrahan, Patricio J.; Rossi, Rolando

doi:10.1074/jbc.m105886200

Cited by 25 publications

(25 citation statements)

References 27 publications

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“…Under this condition, the shared sites may be transiently empty of ions, making the α-subunit more liable to proteolytic attack. This explanation is consistent with the well-established fact that whereas the K + occluded form is formed spontaneously after incubation of the dephosphorylated enzyme with K + /Rb + [45], the Na + occluded form is not observed unless the enzyme is phosphorylated from ATP [46].…”

Section: Discussionsupporting

confidence: 91%

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

et al. 2014

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Capsazepine (CPZ) inhibits Na+,K+-ATPase-mediated K+-dependent ATP hydrolysis with no effect on Na+-ATPase activity. In this study we have investigated the functional effects of CPZ on Na+,K+-ATPase in intact cells. We have also used well established biochemical and biophysical techniques to understand how CPZ modifies the catalytic subunit of Na+,K+-ATPase. In isolated rat cardiomyocytes, CPZ abolished Na+,K+-ATPase current in the presence of extracellular K+. In contrast, CPZ stimulated pump current in the absence of extracellular K+. Similar conclusions were attained using HEK293 cells loaded with the Na+ sensitive dye Asante NaTRIUM green. Proteolytic cleavage of pig kidney Na+,K+-ATPase indicated that CPZ stabilizes ion interaction with the K+ sites. The distal part of membrane span 10 (M10) of the α-subunit was exposed to trypsin cleavage in the presence of guanidinum ions, which function as Na+ congener at the Na+ specific site. This effect of guanidinium was amplified by treatment with CPZ. Fluorescence of the membrane potential sensitive dye, oxonol VI, was measured following addition of substrates to reconstituted inside-out Na+,K+-ATPase. CPZ increased oxonol VI fluorescence in the absence of K+, reflecting increased Na+ efflux through the pump. Surprisingly, CPZ induced an ATP-independent increase in fluorescence in the presence of high extravesicular K+, likely indicating opening of an intracellular pathway selective for K+. As revealed by the recent crystal structure of the E1.AlF4 -.ADP.3Na+ form of the pig kidney Na+,K+-ATPase, movements of M5 of the α-subunit, which regulate ion selectivity, are controlled by the C-terminal tail that extends from M10. We propose that movements of M10 and its cytoplasmic extension is affected by CPZ, thereby regulating ion selectivity and transport through the K+ sites in Na+,K+-ATPase.

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Section: Discussionsupporting

confidence: 91%

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

et al. 2014

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“…ϩ is the only ligand of the Na ϩ /K ϩ -ATPase both the kinetics of direct occlusion and deocclusion and the equilibrium distribution between free and occluded Rb ϩ seem to indicate that either one or two Rb ϩ can be occluded per Na ϩ /K ϩ -ATPase molecule (1). This contrasts with the experimental evidence that under physiological conditions occlusion takes place only when two Rb ϩ are trapped per enzyme molecule.…”

Section: In Media In Which Rbcontrasting

confidence: 52%

“…The enzyme preparation, incubation conditions, reagents, methods for the determination of occluded Rb ϩ as well as the statistical procedures applied to the data are described in the previous paper of this series (1). ATP was purchased as the disodium salt and freed of Na ϩ by passing a 100 mM solution of ATP followed by 1 ml of 200 mM imidazoleHCl (pH 7.4 at 25°C) through a column containing 1 ml of a cation exchange resin (Bio-Rad AG MP-50).…”

Section: Methodsmentioning

confidence: 99%

“…Model Selection-Regression procedures permitted to define the goodness of fit of a given equation to the experimental results and to choose among different models, by using the AIC criterion (2) which, as mentioned in the previous paper (1), is defined as AIC ϭ N ln(SS) ϩ 2 P, with N ϭ number of data, P ϭ number of parameters, and SS ϭ sum of weighted square residual errors. Statistical weights were 1 in all cases.…”

Section: Methodsmentioning

confidence: 99%

“…Since occlusion only takes place in the E 2 conformer, if the E 2 ATP complex were able to bind and occlude Rb ϩ , the occlusion that follows binding would displace the equilibrium between E 1 and E 2 completely toward E 2 . ATP acting on E 2 induces a large increase in the rate of release of occluded Rb ϩ (1,5,17,21,22). Therefore, either this increase is unable to significantly poise the equilibrium between E 1 and E 2 toward E 1 , or the postulate of occlusion in E 2 ATP has to be accompanied by the additional proposal that ATP induces an increase in the rate of formation of occluded Rb ϩ large enough as to keep the maximal amount of Rb occ independent of the concentration of the nucleotide.…”

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confidence: 99%

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The Occlusion of Rb+ in the Na+/K+-ATPase

González-Lebrero¹,

Kaufman

Garrahan³

et al. 2002

Journal of Biological Chemistry

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Mechanism of allosteric effects of ATP on the kinetics of P-type ATPases

Clarke

2009

Eur Biophys J

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The roles of allosteric effects of ATP and protein oligomerisation in the mechanisms of P-type ATPases belong to the most controversial and least well understood topics in the field. Recent crystal structural and kinetic data, however, now allow certain hypotheses to be definitely excluded and consistent hypotheses to be developed. The aim of this review is to critically discuss recent results and, in the light of them, to present a set of conclusions which could form the basis of future research. The major conclusions are: (1) at saturating ATP concentrations P-type ATPases function as monomeric enzymes, (2) the catalytic units of P-type ATPases only possess a single ATP binding site, (3) at non-saturating ATP concentrations P-type ATPases exist as diprotomeric (or higher oligomeric) complexes, (4) protein-protein interactions within a diprotomeric complex enhances the enzymes' ATP binding affinity, (5) ATP binding to both protomers within a diprotomeric complex causes it to dissociate into two separate monomers. The physiological role of protein-protein interactions within a diprotomer may be to enhance ATP binding affinity so as to scavenge ATP and maximize the ion pumping rate under hypoxic or anoxic conditions. For the first time a structural basis for the well-known ATP allosteric acceleration of the E2 --> E1 transition is presented. This is considered to be due to a minimization of steric hindrance between neighbouring protomers because of the ability of ATP to induce a compact conformation of the enzymes' cytoplasmic domains.

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The Occlusion of Rb+ in the Na+/K+-ATPase

Abstract: Occlusion of K؉ or its congeners in the Na ؉ /K ؉ -ATPase occurs after K ؉ -dependent dephosphorylation (physiological route) or in media lacking ATP and Na

Cited by 25 publications

References 27 publications

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

Inhibition of K+ Transport through Na+, K+-ATPase by Capsazepine: Role of Membrane Span 10 of the α-Subunit in the Modulation of Ion Gating

The Occlusion of Rb+ in the Na+/K+-ATPase

Mechanism of allosteric effects of ATP on the kinetics of P-type ATPases

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