Publications by authors named "Bente Vilsen"

58 Publications

ATP1A2- and ATP1A3-associated early profound epileptic encephalopathy and polymicrogyria.

Brain 2021 Apr 21. Epub 2021 Apr 21.

Pediatric Neurology, Neurogenetics and Neurobiology Unit and Laboratories, Meyer Children's Hospital, University of Florence, Florence, Italy.

Constitutional heterozygous mutations of ATP1A2 and ATP1A3, encoding for two distinct isoforms of the Na+/K+-ATPase (NKA) alpha-subunit, have been associated with familial hemiplegic migraine (ATP1A2), alternating hemiplegia of childhood (ATP1A2/A3), rapid-onset dystonia-parkinsonism, cerebellar ataxia-areflexia-progressive optic atrophy, and relapsing encephalopathy with cerebellar ataxia (all ATP1A3). A few reports have described single individuals with heterozygous mutations of ATP1A2/A3 associated with severe childhood epilepsies. Early lethal hydrops fetalis, arthrogryposis, microcephaly, and polymicrogyria have been associated with homozygous truncating mutations in ATP1A2. We investigated the genetic causes of developmental and epileptic encephalopathies variably associated with malformations of cortical development in a large cohort and identified 22 patients with de novo or inherited heterozygous ATP1A2/A3 mutations. We characterized clinical, neuroimaging and neuropathological findings, performed in silico and in vitro assays of the mutations' effects on the NKA-pump function, and studied genotype-phenotype correlations. Twenty-two patients harboured 19 distinct heterozygous mutations of ATP1A2 (six patients, five mutations) and ATP1A3 (16 patients, 14 mutations, including a mosaic individual). Polymicrogyria occurred in 10 (45%) patients, showing a mainly bilateral perisylvian pattern. Most patients manifested early, often neonatal, onset seizures with a multifocal or migrating pattern. A distinctive, 'profound' phenotype, featuring polymicrogyria or progressive brain atrophy and epilepsy, resulted in early lethality in seven patients (32%). In silico evaluation predicted all mutations to be detrimental. We tested 14 mutations in transfected COS-1 cells and demonstrated impaired NKA-pump activity, consistent with severe loss of function. Genotype-phenotype analysis suggested a link between the most severe phenotypes and lack of COS-1 cell survival, and also revealed a wide continuum of severity distributed across mutations that variably impair NKA-pump activity. We performed neuropathological analysis of the whole brain in two individuals with polymicrogyria respectively related to a heterozygous ATP1A3 mutation and a homozygous ATP1A2 mutation and found close similarities with findings suggesting a mainly neural pathogenesis, compounded by vascular and leptomeningeal abnormalities. Combining our report with other studies, we estimate that ∼5% of mutations in ATP1A2 and 12% in ATP1A3 can be associated with the severe and novel phenotypes that we describe here. Notably, a few of these mutations were associated with more than one phenotype. These findings assign novel, 'profound' and early lethal phenotypes of developmental and epileptic encephalopathies and polymicrogyria to the phenotypic spectrum associated with heterozygous ATP1A2/A3 mutations and indicate that severely impaired NKA pump function can disrupt brain morphogenesis.
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http://dx.doi.org/10.1093/brain/awab052DOI Listing
April 2021

Binding of cardiotonic steroids to Na,K-ATPase in the E2P state.

Proc Natl Acad Sci U S A 2021 01;118(1)

Institute for Quantitative Biosciences, The University of Tokyo, 113-0032 Tokyo, Japan;

The sodium pump (Na, K-ATPase, NKA) is vital for animal cells, as it actively maintains Na and K electrochemical gradients across the cell membrane. It is a target of cardiotonic steroids (CTSs) such as ouabain and digoxin. As CTSs are almost unique strong inhibitors specific to NKA, a wide range of derivatives has been developed for potential therapeutic use. Several crystal structures have been published for NKA-CTS complexes, but they fail to explain the largely different inhibitory properties of the various CTSs. For instance, although CTSs are thought to inhibit ATPase activity by binding to NKA in the E2P state, we do not know if large conformational changes accompany binding, as no crystal structure is available for the E2P state free of CTS. Here, we describe crystal structures of the BeF complex of NKA representing the E2P ground state and then eight crystal structures of seven CTSs, including rostafuroxin and istaroxime, two new members under clinical trials, in complex with NKA in the E2P state. The conformations of NKA are virtually identical in all complexes with and without CTSs, showing that CTSs bind to a preformed cavity in NKA. By comparing the inhibitory potency of the CTSs measured under four different conditions, we elucidate how different structural features of the CTSs result in different inhibitory properties. The crystal structures also explain K-antagonism and suggest a route to isoform specific CTSs.
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http://dx.doi.org/10.1073/pnas.2020438118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817145PMC
January 2021

Distinct effects of Q925 mutation on intracellular and extracellular Na and K binding to the Na, K-ATPase.

Sci Rep 2019 09 16;9(1):13344. Epub 2019 Sep 16.

Department of Biomedicine, Aarhus University, DK-8000, Aarhus C, Denmark.

Three Na sites are defined in the Na-bound crystal structure of Na, K-ATPase. Sites I and II overlap with two K sites in the K-bound structure, whereas site III is unique and Na specific. A glutamine in transmembrane helix M8 (Q925) appears from the crystal structures to coordinate Na at site III, but does not contribute to K coordination at sites I and II. Here we address the functional role of Q925 in the various conformational states of Na, K-ATPase by examining the mutants Q925A/G/E/N/L/I/Y. We characterized these mutants both enzymatically and electrophysiologically, thereby revealing their Na and K binding properties. Remarkably, Q925 substitutions had minor effects on Na binding from the intracellular side of the membrane - in fact, mutations Q925A and Q925G increased the apparent Na affinity - but caused dramatic reductions of the binding of K as well as Na from the extracellular side of the membrane. These results provide insight into the changes taking place in the Na-binding sites, when they are transformed from intracellular- to extracellular-facing orientation in relation to the ion translocation process, and demonstrate the interaction between sites III and I and a possible gating function of Q925 in the release of Na at the extracellular side.
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http://dx.doi.org/10.1038/s41598-019-50009-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746705PMC
September 2019

Asparagine 905 of the mammalian phospholipid flippase ATP8A2 is essential for lipid substrate-induced activation of ATP8A2 dephosphorylation.

J Biol Chem 2019 04 13;294(15):5970-5979. Epub 2019 Feb 13.

From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address:

The P-type ATPase protein family includes, in addition to ion pumps such as Ca-ATPase and Na,K-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 ( V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.
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http://dx.doi.org/10.1074/jbc.RA118.007240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463696PMC
April 2019

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

J Biol Chem 2019 01 8;294(1):269-280. Epub 2018 Nov 8.

Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark. Electronic address:

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 EP and EP. 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-E 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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http://dx.doi.org/10.1074/jbc.RA118.004591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322875PMC
January 2019

Germline De Novo Mutations in ATP1A1 Cause Renal Hypomagnesemia, Refractory Seizures, and Intellectual Disability.

Am J Hum Genet 2018 11;103(5):808-816

Department of General Pediatrics, University Children's Hospital, Münster 48149, Germany.

Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na, K-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na, K-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na, K-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na, K-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity.
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http://dx.doi.org/10.1016/j.ajhg.2018.10.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6218849PMC
November 2018

Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates.

Sci Rep 2017 09 5;7(1):10418. Epub 2017 Sep 5.

Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Bldg. 1160, DK-8000, Aarhus C, Denmark.

Phospholipid flippases (P-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the "giant" lipid substrate has not been determined. ATP8A2 is a flippase with selectivity toward phosphatidylserine (PS), possessing a net negatively charged head group, whereas ATP8B1 exhibits selectivity toward the electrically neutral phosphatidylcholine (PC). Setting out to elucidate the functional consequences of flippase disease mutations, we have identified residues of ATP8A2 that are critical to the interaction with the lipid substrate during the translocation process. Among the residues pinpointed are I91 and L308, which are positioned near proposed translocation routes through the protein. In addition we pinpoint two juxtaposed oppositely charged residues, E897 and R898, in the exoplasmic loop between transmembrane helices 5 and 6. The glutamate is conserved between PS and PC flippases, whereas the arginine is replaced by a negatively charged aspartate in ATP8B1. Our mutational analysis suggests that the glutamate repels the PS head group, whereas the arginine minimizes this repulsion in ATP8A2, thereby contributing to control the entry of the phospholipid substrate into the translocation pathway.
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http://dx.doi.org/10.1038/s41598-017-10741-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585164PMC
September 2017

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.

Glia 2017 11 8;65(11):1777-1793. Epub 2017 Aug 8.

Center for Neuroscience, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

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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http://dx.doi.org/10.1002/glia.23194DOI Listing
November 2017

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

Proc Natl Acad Sci U S A 2017 01 27;114(2):316-321. Epub 2016 Dec 27.

Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark;

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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http://dx.doi.org/10.1073/pnas.1617951114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240710PMC
January 2017

Neurological disease mutations of α3 Na,K-ATPase: Structural and functional perspectives and rescue of compromised function.

Biochim Biophys Acta 2016 11 28;1857(11):1807-1828. Epub 2016 Aug 28.

Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark. Electronic address:

Na,K-ATPase creates transmembrane ion gradients crucial to the function of the central nervous system. The α-subunit of Na,K-ATPase exists as four isoforms (α1-α4). Several neurological phenotypes derive from α3 mutations. The effects of some of these mutations on Na,K-ATPase function have been studied in vitro. Here we discuss the α3 disease mutations as well as information derived from studies of corresponding mutations of α1 in the light of the high-resolution crystal structures of the Na,K-ATPase. A high proportion of the α3 disease mutations occur in the transmembrane sector and nearby regions essential to Na and K binding. In several cases the compromised function can be traced to disturbance of the Na specific binding site III. Recently, a secondary mutation was found to rescue the defective Na binding caused by a disease mutation. A perspective is that it may be possible to develop an efficient pharmaceutical mimicking the rescuing effect.
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http://dx.doi.org/10.1016/j.bbabio.2016.08.009DOI Listing
November 2016

Glutamate transporter activity promotes enhanced Na /K -ATPase-mediated extracellular K management during neuronal activity.

J Physiol 2016 11 29;594(22):6627-6641. Epub 2016 Jun 29.

Department Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Key Points: Management of glutamate and K in brain extracellular space is of critical importance to neuronal function. The astrocytic α2β2 Na /K -ATPase isoform combination is activated by the K transients occurring during neuronal activity. In the present study, we report that glutamate transporter-mediated astrocytic Na transients stimulate the Na /K -ATPase and thus the clearance of extracellular K . Specifically, the astrocytic α2β1 Na /K -ATPase subunit combination displays an apparent Na affinity primed to react to physiological changes in intracellular Na . Accordingly, we demonstrate a distinct physiological role in K management for each of the two astrocytic Na /K -ATPase β-subunits.

Abstract: Neuronal activity is associated with transient [K ] increases. The excess K is cleared by surrounding astrocytes, partly by the Na /K -ATPase of which several subunit isoform combinations exist. The astrocytic Na /K -ATPase α2β2 isoform constellation responds directly to increased [K ] but, in addition, Na /K -ATPase-mediated K clearance could be governed by astrocytic [Na ] . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na -coupled glutamate transporters, thereby elevating [Na ] . It thus remains unresolved whether the different Na /K -ATPase isoforms are controlled by [K ] or [Na ] during neuronal activity. Hippocampal slice recordings of stimulus-induced [K ] transients with ion-sensitive microelectrodes revealed reduced Na /K -ATPase-mediated K management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na affinity in intact oocytes and isolated membranes. The Na /K -ATPase was not fully saturated at basal astrocytic [Na ] , irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na /K -ATPase-dependent K clearance was obtained with parallel glutamate transport activity. The astrocytic Na /K -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na ] transients associated with activity-induced glutamate transporter activity.
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http://dx.doi.org/10.1113/JP272531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5108912PMC
November 2016

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

J Biol Chem 2016 May 24;291(20):10934-47. Epub 2016 Mar 24.

From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.

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. The reduction of Na(+) affinity is within the range expected for physiological regulation of the intracellular Na(+) concentration, thus supporting the hypothesis that PKA-mediated phosphorylation of Ser-938 regulates Na(+),K(+)-ATPase activity in vivo Ser-938 is located in the intracellular loop between transmembrane segments M8 and M9. An extended bonding network connects this loop with M10, the C terminus, and the Na(+) binding region. Charged residues Asp-997, Glu-998, Arg-1000, and Lys-1001 in M10, participating in this bonding network, are crucial to Na(+) interaction. Replacement of Arg-1005, also located in the vicinity of Ser-938, with alanine, lysine, methionine, or serine resulted in wild type-like Na(+) and K(+) affinities and catalytic turnover rate. However, when combined with the phosphomimetic mutation S938E only lysine substitution of Arg-1005 was compatible with Na(+),K(+)-ATPase function, and the Na(+) affinity of this double mutant was reduced even more than in single mutant S938E. This result indicates that the positive side chain of Arg-1005 or the lysine substituent plays a mechanistic role as interaction partner of phosphorylated Ser-938, transducing the phosphorylation signal into a reduced affinity of Na(+) site III. 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.
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http://dx.doi.org/10.1074/jbc.M115.701201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4865937PMC
May 2016

Specific mutations in mammalian P4-ATPase ATP8A2 catalytic subunit entail differential glycosylation of the accessory CDC50A subunit.

FEBS Lett 2015 Dec 22;589(24 Pt B):3908-14. Epub 2015 Nov 22.

Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Bldg. 1160, 8000 Aarhus C, Denmark.

P4-ATPases, or flippases, translocate phospholipids between the two leaflets of eukaryotic biological membranes. They are essential to the physiologically crucial phospholipid asymmetry and involved in severe diseases, but their molecular structure and mechanism are still unresolved. Here, we show that in an extensive mutational alanine screening of the mammalian flippase ATP8A2 catalytic subunit, five mutations stand out by leading to reduced glycosylation of the accessory subunit CDC50A. These mutations may disturb the interaction between the subunits.
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http://dx.doi.org/10.1016/j.febslet.2015.11.031DOI Listing
December 2015

Rescue of Na+ affinity in aspartate 928 mutants of Na+,K+-ATPase by secondary mutation of glutamate 314.

J Biol Chem 2015 Apr 24;290(15):9801-11. Epub 2015 Feb 24.

From the Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, DK-8000 Aarhus C, Denmark

The Na(+),K(+)-ATPase binds Na(+) at three transport sites denoted I, II, and III, of which site III is Na(+)-specific and suggested to be the first occupied in the cooperative binding process activating phosphorylation from ATP. Here we demonstrate that the asparagine substitution of the aspartate associated with site III found in patients with rapid-onset dystonia parkinsonism or alternating hemiplegia of childhood causes a dramatic reduction of Na(+) affinity in the α1-, α2-, and α3-isoforms of Na(+),K(+)-ATPase, whereas other substitutions of this aspartate are much less disruptive. This is likely due to interference by the amide function of the asparagine side chain with Na(+)-coordinating residues in site III. Remarkably, the Na(+) affinity of site III aspartate to asparagine and alanine mutants is rescued by second-site mutation of a glutamate in the extracellular part of the fourth transmembrane helix, distant to site III. This gain-of-function mutation works without recovery of the lost cooperativity and selectivity of Na(+) binding and does not affect the E1-E2 conformational equilibrium or the maximum phosphorylation rate. Hence, the rescue of Na(+) affinity is likely intrinsic to the Na(+) binding pocket, and the underlying mechanism could be a tightening of Na(+) binding at Na(+) site II, possibly via movement of transmembrane helix four. The second-site mutation also improves Na(+),K(+) pump function in intact cells. Rescue of Na(+) affinity and Na(+) and K(+) transport by second-site mutation is unique in the history of Na(+),K(+)-ATPase and points to new possibilities for treatment of neurological patients carrying Na(+),K(+)-ATPase mutations.
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http://dx.doi.org/10.1074/jbc.M114.625509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392278PMC
April 2015

Distinct neurological disorders with ATP1A3 mutations.

Lancet Neurol 2014 May;13(5):503-14

Department of Biomedicine, Aarhus University, Aarhus, Denmark.

Genetic research has shown that mutations that modify the protein-coding sequence of ATP1A3, the gene encoding the α3 subunit of Na(+)/K(+)-ATPase, cause both rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood. These discoveries link two clinically distinct neurological diseases to the same gene, however, ATP1A3 mutations are, with one exception, disease-specific. Although the exact mechanism of how these mutations lead to disease is still unknown, much knowledge has been gained about functional consequences of ATP1A3 mutations using a range of in-vitro and animal model systems, and the role of Na(+)/K(+)-ATPases in the brain. Researchers and clinicians are attempting to further characterise neurological manifestations associated with mutations in ATP1A3, and to build on the existing molecular knowledge to understand how specific mutations can lead to different diseases.
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http://dx.doi.org/10.1016/S1474-4422(14)70011-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238309PMC
May 2014

Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2.

Proc Natl Acad Sci U S A 2014 Apr 24;111(14):E1334-43. Epub 2014 Mar 24.

Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.

P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.
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http://dx.doi.org/10.1073/pnas.1321165111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986137PMC
April 2014

Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease.

J Biol Chem 2014 Feb 19;289(6):3186-97. Epub 2013 Dec 19.

From the Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark and.

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na(+),K(+)-ATPase α2 and α3 isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na(+),K(+)-ATPase mutations, extending the C terminus by either 28 residues ("+28" mutation) or an extra tyrosine ("+Y"), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na(+) and K(+) concentrations ([Na(+)]i and [K(+)]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na(+) affinity without disturbance of K(+) binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α2 despite a high expression level. A significant rise of [Na(+)]i and reduction of [K(+)]i was detected in cells expressing mutants with reduced Na(+) affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na(+) affinity were found to reduce [Na(+)]i. It is concluded that the Na(+) affinity of the Na(+),K(+)-ATPase is an important determinant of [Na(+)]i.
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http://dx.doi.org/10.1074/jbc.M113.543272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3916523PMC
February 2014

Molecular cloning and characterization of porcine Na⁺/K⁺-ATPase isoforms α1, α2, α3 and the ATP1A3 promoter.

PLoS One 2013 13;8(11):e79127. Epub 2013 Nov 13.

Department of Molecular Biology and Genetics, Aarhus University, Tjele, Denmark ; Department of Biomedicine, Aarhus University, Aarhus C, Denmark.

Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0079127PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3827302PMC
September 2014

Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state.

Nature 2013 Oct 2;502(7470):201-6. Epub 2013 Oct 2.

Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.

Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.
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http://dx.doi.org/10.1038/nature12578DOI Listing
October 2013

Somatic ATP1A1, ATP2B3, and KCNJ5 mutations in aldosterone-producing adenomas.

Hypertension 2014 Jan 30;63(1):188-95. Epub 2013 Sep 30.

Division of Internal Medicine and Hypertension, Department of Medical Sciences, University of Torino, Torino, Italy.

Aldosterone-producing adenomas (APAs) cause a sporadic form of primary aldosteronism and somatic mutations in the KCNJ5 gene, which encodes the G-protein-activated inward rectifier K(+) channel 4, GIRK4, account for ≈40% of APAs. Additional somatic APA mutations were identified recently in 2 other genes, ATP1A1 and ATP2B3, encoding Na(+)/K(+)-ATPase 1 and Ca(2+)-ATPase 3, respectively, at a combined prevalence of 6.8%. We have screened 112 APAs for mutations in known hotspots for genetic alterations associated with primary aldosteronism. Somatic mutations in ATP1A1, ATP2B3, and KCNJ5 were present in 6.3%, 0.9%, and 39.3% of APAs, respectively, and included 2 novel mutations (Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg). CYP11B2 gene expression was higher in APAs harboring ATP1A1 and ATP2B3 mutations compared with those without these or KCNJ5 mutations. Overexpression of Na(+)/K(+)-ATPase p.Gly99Arg and GIRK4 p.Trp126Arg in HAC15 adrenal cells resulted in upregulation of CYP11B2 gene expression and its transcriptional regulator NR4A2. Structural modeling of the Na(+)/K(+)-ATPase showed that the Gly99Arg substitution most likely interferes with the gateway to the ion binding pocket. In vitro functional assays demonstrated that Gly99Arg displays severely impaired ATPase activity, a reduced apparent affinity for Na(+) activation of phosphorylation and K(+) inhibition of phosphorylation that indicate decreased Na(+) and K(+) binding, respectively. Moreover, whole cell patch-clamp studies established that overexpression of Na(+)/K(+)-ATPase Gly99Arg causes membrane voltage depolarization. In conclusion, somatic mutations are common in APAs that result in an increase in CYP11B2 gene expression and may account for the dysregulated aldosterone production in a subset of patients with sporadic primary aldosteronism.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.113.01733DOI Listing
January 2014

Somatic mutations in ATP1A1 and ATP2B3 lead to aldosterone-producing adenomas and secondary hypertension.

Nat Genet 2013 Apr 17;45(4):440-4, 444e1-2. Epub 2013 Feb 17.

Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität München, Munich, Germany.

Primary aldosteronism is the most prevalent form of secondary hypertension. To explore molecular mechanisms of autonomous aldosterone secretion, we performed exome sequencing of aldosterone-producing adenomas (APAs). We identified somatic hotspot mutations in the ATP1A1 (encoding an Na(+)/K(+) ATPase α subunit) and ATP2B3 (encoding a Ca(2+) ATPase) genes in three and two of the nine APAs, respectively. These ATPases are expressed in adrenal cells and control sodium, potassium and calcium ion homeostasis. Functional in vitro studies of ATP1A1 mutants showed loss of pump activity and strongly reduced affinity for potassium. Electrophysiological ex vivo studies on primary adrenal adenoma cells provided further evidence for inappropriate depolarization of cells with ATPase alterations. In a collection of 308 APAs, we found 16 (5.2%) somatic mutations in ATP1A1 and 5 (1.6%) in ATP2B3. Mutation-positive cases showed male dominance, increased plasma aldosterone concentrations and lower potassium concentrations compared with mutation-negative cases. In summary, dominant somatic alterations in two members of the ATPase gene family result in autonomous aldosterone secretion.
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http://dx.doi.org/10.1038/ng.2550DOI Listing
April 2013

Critical role of a transmembrane lysine in aminophospholipid transport by mammalian photoreceptor P4-ATPase ATP8A2.

Proc Natl Acad Sci U S A 2012 Jan 17;109(5):1449-54. Epub 2012 Jan 17.

Departments of Biochemistry and Molecular Biology, and Ophthalmology and Visual Sciences, Centre for Macular Research, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.

ATP8A2 is a P(4)-ATPase ("flippase") located in membranes of retinal photoreceptors, brain cells, and testis, where it mediates transport of aminophospholipids toward the cytoplasmic leaflet. It has long been an enigma whether the mechanism of P(4)-ATPases resembles that of the well-characterized cation-transporting P-type ATPases, and it is unknown whether the flippases interact directly with the lipid and with counterions. Our results demonstrate that ATP8A2 forms a phosphoenzyme intermediate at the conserved aspartate (Asp(416)) in the P-type ATPase signature sequence and exists in E(1)P and E(2)P forms similar to the archetypical P-type ATPases. Using the properties of the phosphoenzyme, the partial reaction steps of the transport cycle were examined, and the roles of conserved residues Asp(196), Glu(198), Lys(873), and Asn(874) in the transport mechanism were elucidated. The former two residues in the A-domain T/D-G-E-S/T motif are involved in catalysis of E(2)P dephosphorylation, the glutamate being essential. Transported aminophospholipids activate the dephosphorylation similar to K(+) activation of dephosphorylation in Na(+),K(+)-ATPase. Lys(873) mutants (particularly K873A and K873E) display a markedly reduced sensitivity to aminophospholipids. Hence, Lys(873), located in transmembrane segment M5 at a "hot spot" for cation binding in Ca(2+)-ATPase and Na(+),K(+)-ATPase, appears to participate directly in aminophospholipid binding or to mediate a crucial interaction within the ATP8A2-CDC50 complex. By contrast, Lys(865) is unimportant for aminophospholipid sensitivity. Binding of Na(+), H(+), K(+), Cl(-), or Ca(2+) to the E(1) form as a counterion is not required for activation of phosphorylation from ATP. Therefore, phospholipids could be the only substrate transported by ATP8A2.
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http://dx.doi.org/10.1073/pnas.1108862109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3277108PMC
January 2012

Inhibition of phosphorylation of na+,k+-ATPase by mutations causing familial hemiplegic migraine.

J Biol Chem 2012 Jan 23;287(3):2191-202. Epub 2011 Nov 23.

Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark.

The neurological disorder familial hemiplegic migraine type II (FHM2) is caused by mutations in the α2-isoform of the Na(+),K(+)-ATPase. We have studied the partial reaction steps of the Na(+),K(+)-pump cycle in nine FHM2 mutants retaining overall activity at a level still compatible with cell growth. Although it is believed that the pathophysiology of FHM2 results from reduced extracellular K(+) clearance and/or changes in Na(+) gradient-dependent transport processes in neuroglia, a reduced affinity for K(+) or Na(+) is not a general finding with the FHM2 mutants. Six of the FHM2 mutations markedly affect the maximal rate of phosphorylation from ATP leading to inhibition by intracellular K(+), thereby likely compromising pump function under physiological conditions. In mutants R593W, V628M, and M731T, the defective phosphorylation is caused by local perturbations within the Rossmann fold, possibly interfering with the bending of the P-domain during phosphoryl transfer. In mutants V138A, T345A, and R834Q, long range effects reaching from as far away as the M2 transmembrane helix perturb the function of the catalytic site. Mutant E700K exhibits a reduced rate of E(2)P dephosphorylation without effect on phosphorylation from ATP. An extremely reduced vanadate affinity of this mutant indicates that the slow dephosphorylation reflects a destabilization of the phosphoryl transition state. This seems to be caused by insertion of the lysine between two other positively charged residues of the Rossmann fold. In mutants R202Q and T263M, effects on the A-domain structure are responsible for a reduced rate of the E(1)P to E(2)P transition.
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http://dx.doi.org/10.1074/jbc.M111.323022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265897PMC
January 2012

Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase α3 sodium pump.

Proc Natl Acad Sci U S A 2011 Nov 24;108(44):18144-9. Epub 2011 Oct 24.

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5.

Bipolar disorder is a debilitating psychopathology with unknown etiology. Accumulating evidence suggests the possible involvement of Na(+),K(+)-ATPase dysfunction in the pathophysiology of bipolar disorder. Here we show that Myshkin mice carrying an inactivating mutation in the neuron-specific Na(+),K(+)-ATPase α3 subunit display a behavioral profile remarkably similar to bipolar patients in the manic state. Myshkin mice show increased Ca(2+) signaling in cultured cortical neurons and phospho-activation of extracellular signal regulated kinase (ERK) and Akt in the hippocampus. The mood-stabilizing drugs lithium and valproic acid, specific ERK inhibitor SL327, rostafuroxin, and transgenic expression of a functional Na(+),K(+)-ATPase α3 protein rescue the mania-like phenotype of Myshkin mice. These findings establish Myshkin mice as a unique model of mania, reveal an important role for Na(+),K(+)-ATPase α3 in the control of mania-like behavior, and identify Na(+),K(+)-ATPase α3, its physiological regulators and downstream signal transduction pathways as putative targets for the design of new antimanic therapies.
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http://dx.doi.org/10.1073/pnas.1108416108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207708PMC
November 2011

A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps.

Nat Rev Mol Cell Biol 2011 Jan;12(1):60-70

Danish National Research Foundation, Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Denmark.

Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.
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http://dx.doi.org/10.1038/nrm3031DOI Listing
January 2011

The rapid-onset dystonia parkinsonism mutation D923N of the Na+, K+-ATPase alpha3 isoform disrupts Na+ interaction at the third Na+ site.

J Biol Chem 2010 Aug 24;285(34):26245-54. Epub 2010 Jun 24.

Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Foundation, Department of Physiology and Biophysics, Aarhus University, DK-8000 Aarhus C, Denmark.

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific alpha3-isoform of Na(+), K(+)-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable approximately 200-fold reduction of Na(+) affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E(1) form with intracellular Na(+). This is the largest effect on Na(+) affinity reported so far for any Na(+), K(+)-ATPase mutant. D923N also affects the interaction with extracellular Na(+) normally driving the E(1)P to E(2)P conformational transition backward. However, no impairment of K(+) binding was observed for D923N, leading to the conclusion that Asp(923) is specifically associated with the third Na(+) site that is selective toward Na(+). The crystal structure of the Na(+), K(+)-ATPase in E(2) form shows that Asp(923) is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na(+) ion. Structural modeling of the E(1) form of Na(+), K(+)-ATPase based on the Ca(2+)-ATPase crystal structure is consistent with the hypothesis that Asp(923) contributes to a site binding the third Na(+) ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na(+) may be a general feature of the RDP disorder.
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http://dx.doi.org/10.1074/jbc.M110.123976DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924038PMC
August 2010

Mutation I810N in the alpha3 isoform of Na+,K+-ATPase causes impairments in the sodium pump and hyperexcitability in the CNS.

Proc Natl Acad Sci U S A 2009 Aug 3;106(33):14085-90. Epub 2009 Aug 3.

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada M5G 1X5.

In a mouse mutagenesis screen, we isolated a mutant, Myshkin (Myk), with autosomal dominant complex partial and secondarily generalized seizures, a greatly reduced threshold for hippocampal seizures in vitro, posttetanic hyperexcitability of the CA3-CA1 hippocampal pathway, and neuronal degeneration in the hippocampus. Positional cloning and functional analysis revealed that Myk/+ mice carry a mutation (I810N) which renders the normally expressed Na(+),K(+)-ATPase alpha3 isoform inactive. Total Na(+),K(+)-ATPase activity was reduced by 42% in Myk/+ brain. The epilepsy in Myk/+ mice and in vitro hyperexcitability could be prevented by delivery of additional copies of wild-type Na(+),K(+)-ATPase alpha3 by transgenesis, which also rescued Na(+),K(+)-ATPase activity. Our findings reveal the functional significance of the Na(+),K(+)-ATPase alpha3 isoform in the control of epileptiform activity and seizure behavior.
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http://dx.doi.org/10.1073/pnas.0904817106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2729024PMC
August 2009

The C terminus of Na+,K+-ATPase controls Na+ affinity on both sides of the membrane through Arg935.

J Biol Chem 2009 Jul 5;284(28):18715-25. Epub 2009 May 5.

Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Aarhus University, DK-8000 Aarhus C, Denmark.

The Na(+),K(+)-ATPase C terminus has a unique location between transmembrane segments, appearing to participate in a network of interactions. We have examined the functional consequences of amino acid substitutions in this region and deletions of the C terminus of varying lengths. Assays revealing separately the mutational effects on internally and externally facing Na(+) sites, as well as E(1)-E(2) conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr(1017) and Tyr(1018), as well as putative interaction partners, Arg(935) in the loop between transmembrane segments M8 and M9 and Lys(768) in transmembrane segment M5, as crucial to Na(+) activation of phosphorylation of E(1), a partial reaction reflecting Na(+) interaction on the cytoplasmic side of the membrane. Tyr(1017), Tyr(1018), and Arg(935) are furthermore indispensable to Na(+) interaction on the extracellular side of the membrane, as revealed by inability of high Na(+) concentrations to drive the transition from E(1)P to E(2)P backwards toward E(1)P and inhibit Na(+)-ATPase activity in mutants. Lys(768) is not important for Na(+) binding from the external side of the membrane but is involved in stabilization of the E(2) form. These data demonstrate that the C terminus controls Na(+) affinity on both sides of the membrane and suggest that Arg(935) constitutes an important link between the C terminus and the third Na(+) site, involving an arginine-pi stacking interaction between Arg(935) and the C-terminal tyrosines. Lys(768) may interact preferentially with the C terminus in E(1) and E(1)P forms and with the loop between transmembrane segments M6 and M7 in E(2) and E(2)P forms.
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http://dx.doi.org/10.1074/jbc.M109.015099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2707196PMC
July 2009

P-type ATPases as drug targets: tools for medicine and science.

Biochim Biophys Acta 2009 Apr;1787(4):207-20

Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Denmark.

P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.
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http://dx.doi.org/10.1016/j.bbabio.2008.12.019DOI Listing
April 2009

A C-terminal mutation of ATP1A3 underscores the crucial role of sodium affinity in the pathophysiology of rapid-onset dystonia-parkinsonism.

Hum Mol Genet 2009 Jul 7;18(13):2370-7. Epub 2009 Apr 7.

Universidad de Santiago de Compostela, Santiago de Compostela, Spain.

The Na(+)/K(+)-ATPases are ion pumps of fundamental importance in maintaining the electrochemical gradient essential for neuronal survival and function. Mutations in ATP1A3 encoding the alpha3 isoform cause rapid-onset dystonia-parkinsonism (RDP). We report a de novo ATP1A3 mutation in a patient with typical RDP, consisting of an in-frame insertion of a tyrosine residue at the very C terminus of the Na(+)/K(+)-ATPase alpha3-subunit-the first reported RDP mutation in the C terminus of the protein. Expression studies revealed that there is no defect in the biogenesis or plasma membrane targeting, although cells expressing the mutant protein showed decreased survival in response to ouabain challenge. Functional analysis demonstrated a drastic reduction in Na(+) affinity in the mutant, which can be understood by structural modelling of the E1 and E2 conformations of the wild-type and mutant enzymes on the basis of the strategic location of the C terminus in relation to the third Na(+) binding site. The dramatic clinical presentation, together with the biochemical findings, provides both in vivo and in vitro evidence for a crucial role of the C terminus of the alpha-subunit in the function of the Na(+)/K(+)-ATPase and a key impact of Na(+) affinity in the pathophysiology of RDP.
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http://dx.doi.org/10.1093/hmg/ddp170DOI Listing
July 2009