Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase

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Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase. / Roenn, Christian P; Li, Melody; Schack, Vivien R et al.

I: Journal of Biological Chemistry, Bind 294, Nr. 1, 04.01.2019, s. 269-280.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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Roenn, Christian P et al. "Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase". Journal of Biological Chemistry. 2019, 294(1). 269-280. https://doi.org/10.1074/jbc.RA118.004591

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Roenn, Christian P ; Li, Melody ; Schack, Vivien R et al. / Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase. I: Journal of Biological Chemistry. 2019 ; Bind 294, Nr. 1. s. 269-280.

Bibtex

@article{15eb2f32d11d4d938c96a6a8571bce60,
title = "Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase",
abstract = "The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.",
keywords = "Adenosine Triphosphate/genetics, Amino Acid Substitution, Animals, Cerebellar Ataxia/genetics, Foot Deformities, Congenital/genetics, Hearing Loss, Sensorineural/genetics, Humans, Membrane Potentials, Mutation, Missense, Optic Atrophy/genetics, Protein Domains, Reflex, Abnormal/genetics, Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors, Vanadates/pharmacology, Xenopus laevis",
author = "Roenn, {Christian P} and Melody Li and Schack, {Vivien R} and Forster, {Ian C} and Rikke Holm and Toustrup-Jensen, {Mads S} and Andersen, {Jens P} and Steven Petrou and Bente Vilsen",
year = "2019",
month = jan,
day = "4",
doi = "10.1074/jbc.RA118.004591",
language = "English",
volume = "294",
pages = "269--280",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "1",

}

RIS

TY - JOUR

T1 - Functional consequences of the CAPOS mutation E818K of Na+,K+-ATPase

AU - Roenn, Christian P

AU - Li, Melody

AU - Schack, Vivien R

AU - Forster, Ian C

AU - Holm, Rikke

AU - Toustrup-Jensen, Mads S

AU - Andersen, Jens P

AU - Petrou, Steven

AU - Vilsen, Bente

PY - 2019/1/4

Y1 - 2019/1/4

N2 - The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

AB - The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the α3-isoform of Na+,K+-ATPase. Here, using biochemical and electrophysiological approaches, we examined the functional characteristics of E818K, as well as of E818Q and E818A mutants. We found that these amino acid substitutions reduce the apparent Na+ affinity at the cytoplasmic-facing sites of the pump protein and that this effect is more pronounced for the lysine and glutamine substitutions (3-4-fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ∼30-fold reduction of apparent Na+ affinity for the extracellular-facing sites in the CAPOS mutant, which was related to an accelerated transition between the phosphoenzyme intermediates E1P and E2P. The apparent affinity for K+ activation of the ATPase activity was unaffected by these substitutions, suggesting that primarily the Na+-specific site III is affected. Furthermore, the apparent affinities for ATP and vanadate were WT-like in E818K, indicating a normal E1-E2 equilibrium of the dephosphoenzyme. Proton-leak currents were not increased in E818K. However, the CAPOS mutation caused a weaker voltage dependence of the pumping rate and a stronger inhibition by cytoplasmic K+ than the WT enzyme, which together with the reduced Na+ affinity of the cytoplasmic-facing sites precluded proper pump activation under physiological conditions. The functional deficiencies could be traced to the participation of Glu-818 in an intricate hydrogen-bonding/salt-bridge network, connecting it to key residues involved in Na+ interaction at site III.

KW - Adenosine Triphosphate/genetics

KW - Amino Acid Substitution

KW - Animals

KW - Cerebellar Ataxia/genetics

KW - Foot Deformities, Congenital/genetics

KW - Hearing Loss, Sensorineural/genetics

KW - Humans

KW - Membrane Potentials

KW - Mutation, Missense

KW - Optic Atrophy/genetics

KW - Protein Domains

KW - Reflex, Abnormal/genetics

KW - Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors

KW - Vanadates/pharmacology

KW - Xenopus laevis

U2 - 10.1074/jbc.RA118.004591

DO - 10.1074/jbc.RA118.004591

M3 - Journal article

C2 - 30409907

VL - 294

SP - 269

EP - 280

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 1

ER -