Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

Toustrup-Jensen, Mads S.; Einholm, Anja P.; Schack, Vivien Rodacker; Nielsen, Henning Κ.; Holm, Rikke; Sobrido, María‐Jesús; Andersen, Jens Peter; Clausen, Torben; Vilsen, Bente

doi:10.1074/jbc.m113.543272

Cited by 40 publications

(45 citation statements)

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“…To determine whether the mutations affect the K + transport steps of the Na + ,K + -pump cycle, we measured active accumulation of K + in COS-1 cells expressing C932R, C932A, or wild-type Na + ,K + -ATPase, using 86 Rb + as a K + congener (14,22). The results obtained following correction for variance in protein expression show little or no difference in K + uptake rates between the mutants and wildtype pumps (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Recently, the homologous replacement of C927 of the α3-isoform of Na + ,K + -ATPase with phenylalanine, tyrosine, or tryptophan was identified in patients with the neurological disease alternating hemiplegia of childhood (11). The very low Na + affinity likely results in a rise of the intracellular Na + concentration that contributes to the pathophysiology (22).…”

Section: Discussionmentioning

confidence: 99%

“…Mutants and wild type were expressed in COS-1 cells under ouabain selection pressure (13,22). Studies of enzyme function were performed on isolated plasma membranes made leaky to allow access of incubation media from both sides of the membrane.…”

Section: Methodsmentioning

confidence: 99%

“…The active site concentration was determined as the maximum phosphorylation from [γ-32 P]ATP measured in the presence of 150 mM NaCl and oligomycin. Cellular K + transport at 37°C was determined using the K + congener 86 Rb + (22), and the uptake data were normalized to represent identical expression levels as calculated from the active site concentration.…”

Section: Methodsmentioning

confidence: 99%

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Holm

Khandelwal

Einholm

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Na+,K+-ATPase and H+,K+-ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H+,K+-ATPase avoids binding of Na+ at the site corresponding to the Na+-specific site of the Na+,K+-ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na+,K+-ATPase with arginine, present in the H+,K+-ATPase at the corresponding position, converted the normal 3Na+:2K+:1ATP stoichiometry of the Na+,K+-ATPase to electroneutral 2Na+:2K+:1ATP stoichiometry similar to the electroneutral transport mode of the H+,K+-ATPase. The electroneutral C932R mutant of the Na+,K+-ATPase retained a wild-type–like enzyme turnover rate for ATP hydrolysis and rate of cellular K+ uptake. Only a relatively minor reduction of apparent Na+ affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na+ affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine+ group of the M8 arginine replaces Na+ at the third site, thus preventing Na+ binding there, although allowing Na+ to bind at the two other sites and become transported. Hence, in the H+,K+-ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Holm

Khandelwal

Einholm

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…To confirm the stable integration of the cDNA carrying the intended single or double mutations, the genomic DNA was isolated, and the integrated cDNA was amplified by PCR using primers that span exon-exon boundaries as described previously (22). In a few cases where the expressed exogenous mutant Na ϩ ,K ϩ -ATPase did not support cell growth in the presence of ouabain, transient co-expression with siRNA to knock down the endogenous COS-1 Na ϩ ,K ϩ -ATPase was carried out as described previously (23,24).…”

Section: Methodsmentioning

confidence: 99%

Importance of a Potential Protein Kinase A Phosphorylation Site of Na+,K+-ATPase and Its Interaction Network for Na+ Binding

Einholm

Nielsen

Holm

et al. 2016

Journal of Biological Chemistry

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The molecular mechanism underlying PKA-mediated regulation of Na ؉ ,K ؉ -ATPase was explored in mutagenesis studies of the potential PKA site at Ser-938 and surrounding charged residues. The phosphomimetic mutations S938D/E interfered with Na ؉ binding from the intracellular side of the membrane, whereas Na ؉ binding from the extracellular side was unaffected. Electrostatic interaction of Glu-998 is of minor importance for the reduction of Na ؉ affinity by phosphomimetic S938E as revealed by combining S938E with E998A.ATPase is a plasma membrane-associated ion pump protein that actively extrudes three Na ϩ ions from the cell while importing two K ϩ ions for each ATP being hydrolyzed (1-4). It is found in all animal cells where its main function is to establish and maintain transmembrane Na ϩ and K ϩ gradients that are fundamental to a variety of physiological processes ranging from electrical excitability of neurons in the brain to Na ϩ reabsorption in the kidney. The Na ϩ ,K ϩ -ATPase belongs to the superfamily of P-type ATPases and is a heterooligomeric protein consisting of a catalytic ␣-subunit, a ␤-subunit required for translocation of the protein complex to the plasma membrane, and a small regulatory protein belonging to the FXYD family (for reviews, see Refs. 4 and 5). The ␣-subunit is made up of a membrane part containing transmembrane helices 1-10 (M1-M10) 2 and a cytoplasmic "head" formed from three subdomains: actuator (A), nucleotide binding (N), and phosphorylation (P) domains (Fig. 1B). During the Na ϩ ,K ϩ -pump cycle (reaction scheme in Fig. 1A), ATP binds to the Nand P-domains, and the ␥-phosphate is transferred to a conserved aspartic acid residue of the P-domain. This phosphorylation is triggered by Na ϩ binding from the cytoplasmic side of the membrane, whereas dephosphorylation with release of inorganic phosphate is triggered by K ϩ binding from the extracellular side. The Na ϩ and K ϩ binding residues are found in the transmembrane helices M4, M5, M6, and M8 (6 -8). Na ϩ ,K ϩ -ATPase alternates between so-called E 1 and E 2 states (1, 2) (see reaction scheme in Fig. 1A). E 1 states bind Na ϩ at three sites denoted I, II, and III, whereas E 2 states prefer K ϩ , binding at two sites, I and II, that overlap considerably with Na ϩ sites I and II. The Na ϩ selectivity of site III is thought to arise in part from interactions of the two C-terminal tyrosines, stabilizing the position of the M5 helix, such that Na ϩ , but not the larger K ϩ ion, can fit into site III, which is the first of the three Na ϩ sites to be occupied when Na ϩ binds from the intracellular side (6,8,9). In addition to the autophosphorylation of the P-domain conserved aspartate, which is central to the reaction cycle of Na ϩ ,K ϩ -ATPase, the Na ϩ ,K ϩ -ATPase activity depends on regulatory phosphorylation by protein kinases (for a review, see Ref. 10). In cardiac myocytes, "kinase-mediated" phosphorylation of the FXYD1 protein stimulates Na ϩ ,K ϩ -ATPase (11). Furthermore, phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit...

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The α2β2 isoform combination dominates the astrocytic Na⁺/K⁺-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

Stoica

Larsen

Assentoft

et al. 2017

Glia

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Synaptic activity results in transient elevations in extracellular K , clearance of which is critical for sustained function of the nervous system. The K clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na /K -ATPase. The Na /K -ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi-quantification was carried out with polyhistidine-tagged Na /K -ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi-quantification of the astrocytic fraction indicated that the astrocytic Na /K -ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine-associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2 mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K dynamics.

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Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

Abstract: Background: Na ϩ ,K ϩ -ATPase mutations extending the C terminus cause neurological disease.

Cited by 40 publications

References 45 publications

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Importance of a Potential Protein Kinase A Phosphorylation Site of Na+,K+-ATPase and Its Interaction Network for Na+ Binding

The α2β2 isoform combination dominates the astrocytic Na⁺/K⁺-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

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Relationship between Intracellular Na+ Concentration and Reduced Na+ Affinity in Na+,K+-ATPase Mutants Causing Neurological Disease

Abstract: Background: Na ϩ ,K ϩ -ATPase mutations extending the C terminus cause neurological disease.

Cited by 40 publications

References 45 publications

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

Arginine substitution of a cysteine in transmembrane helix M8 converts Na + ,K + -ATPase to an electroneutral pump similar to H + ,K + -ATPase

Importance of a Potential Protein Kinase A Phosphorylation Site of Na+,K+-ATPase and Its Interaction Network for Na+ Binding

The α2β2 isoform combination dominates the astrocytic Na+/K+-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

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Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

Arginine substitution of a cysteine in transmembrane helix M8 converts Na ⁺ ,K ⁺ -ATPase to an electroneutral pump similar to H ⁺ ,K ⁺ -ATPase

The α2β2 isoform combination dominates the astrocytic Na⁺/K⁺-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation