Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

Holm, Rikke; Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

doi:10.1074/jbc.m114.625509

Cited by 14 publications

(14 citation statements)

References 25 publications

(14 reference statements)

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“…This seems to be in accord with the free energy perturbation calculations done by Rui et al (14) demonstrating that the charge neutralizing the D926N mutation eliminates Na ϩ selectivity. In fact, D926N mutants have a decreased apparent Na ϩ affinity, despite a slight E1/E2 conformational shift toward E1 (33). For the outward-facing K ϩ sites, pK a estimations suggest that increasing pH results in deprotonation of Asp 926 and perhaps Glu 327 .…”

Section: Discussionmentioning

confidence: 99%

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase

Cornelius

Tsunekawa

Toyoshima

2018

Journal of Biological Chemistry

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The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis-powered Na and K electrochemical gradients across the cell membrane. These ion gradients drive co- and countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na and K, despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2·Pi·2K and Na-bound E1∼P·ADP suggest that the dimensions of the respective binding sites in Na,K-ATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K at the extracellular face ( E2 conformation) as the pH is lowered from 7.5 to 5. We also observed a smaller increase in affinity for K on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na Theoretical analysis of the p values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na/K selectivities may arise from the protonation or ionization of key residues. The increase in K selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity.

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Section: Discussionmentioning

confidence: 99%

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase

Cornelius

Tsunekawa

Toyoshima

2018

Journal of Biological Chemistry

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“…Measurement of characteristic leak current with mutations in site III revealed that the Na + binding affinity in Asp926 mutants are less sensitive to extracellular sodium, whereas Glu954 mutations cause less leakage57. Although a recent study suggests that Asp926 coordinates site III, the role of Asp926 is described as “relatively minor” in mutagenesis experiments58. However, our six best candidates for the most stable Na + bound protonation states suggest that at least one proton should be available to each Na + binding site.…”

Section: Resultsmentioning

confidence: 99%

“…The water molecule movement through each pathway was calculated as described in the Supporting Information. There are three water pathways in the NKA, including the contentious C-terminal pathway1458, and each pathway is closely related to the acidic residues around each binding site. The extracellular pathway is near site I and two intracellular pathways, N- and C-terminal pathway are near site II and site III, respectively1214 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Glutamate Water Gates in the Ion Binding Pocket of Na+ Bound Na+, K+-ATPase

Han

Kopeć

Solov’yov

et al. 2017

Sci Rep

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The dynamically changing protonation states of the six acidic amino acid residues in the ion binding pocket of the Na+, K+ -ATPase (NKA) during the ion transport cycle are proposed to drive ion binding, release and possibly determine Na+ or K+ selectivity. We use molecular dynamics (MD) and density functional theory (DFT) simulations to determine the protonation scheme of the Na+ bound conformation of NKA. MD simulations of all possible protonation schemes show that the bound Na+ ions are most stably bound when three or four protons reside in the binding sites, and that Glu954 in site III is always protonated. Glutamic acid residues in the three binding sites act as water gates, and their deprotonation triggers water entry to the binding sites. From DFT calculations of Na+ binding energies, we conclude that three protons in the binding site are needed to effectively bind Na+ from water and four are needed to release them in the next step. Protonation of Asp926 in site III will induce Na+ release, and Glu327, Glu954 and Glu779 are all likely to be protonated in the Na+ bound occluded conformation. Our data provides key insights into the role of protons in the Na+ binding and release mechanism of NKA.

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“…The SigmaPlot program (SPSS, Inc.) was used to fit the relevant equations to data points using nonlinear regression (48). The Na ϩ and ATP dependences of the ATPase activity or phosphorylation were fitted by the following Hill function.…”

Section: Biochemical Experimentsmentioning

confidence: 99%

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

Roenn

Schack

et al. 2019

Journal of Biological Chemistry

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The cerebellar ataxia, areflexia, pes cavus, optic atrophy, and sensorineural hearing loss (CAPOS) syndrome is caused by the single mutation E818K of the ␣3-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 (3-4fold) than for the alanine substitution. The electrophysiological measurements indicated a more conspicuous, ϳ30-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 E 1 P and E 2 P. 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 E 1 -E 2 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.

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Rescue of Na+ Affinity in Aspartate 928 Mutants of Na+,K+-ATPase by Secondary Mutation of Glutamate 314

Abstract: Background: Mutation of the aspartate associated with Na ϩ site III of Na ϩ ,K ϩ -ATPase causes neurological disorders.

Cited by 14 publications

References 25 publications

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase

Glutamate Water Gates in the Ion Binding Pocket of Na+ Bound Na+, K+-ATPase

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

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