Publications by authors named "Rikke Holm"

16 Publications

  • Page 1 of 1

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

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

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

Quality of care for people with multimorbidity - a case series.

BMC Health Serv Res 2017 Nov 18;17(1):745. Epub 2017 Nov 18.

Research Unit for Chronic Conditions, Department of Clinical Epidemiology, Bispebjerg and Frederiksberg Hospitals, University of Copenhagen, Copenhagen, Denmark.

Background: Multimorbidity is becoming increasingly prevalent and presents challenges for healthcare providers and systems. Studies examining the relationship between multimorbidity and quality of care report mixed findings. The purpose of this study was to investigate quality of care for people with multimorbidity in the publicly funded healthcare system in Denmark.

Methods: To investigate the quality of care for people with multimorbidity different groups of clinicians from the hospital, general practice and the municipality reviewed records from 23 persons with multimorbidity and discussed them in three focus groups. Before each focus group, clinicians were asked to review patients' medical records and assess their care by responding to a questionnaire. Medical records from 2013 from hospitals, general practice, and health centers in the local municipality were collected and linked for the 23 patients. Further, two clinical pharmacologists reviewed the appropriateness of medications listed in patient records.

Results: The review of the patients' records conducted by three groups of clinicians revealed that around half of the patients received adequate care for the single condition which prompted the episode of care such as a hospitalization, a visit to an outpatient clinic or the general practitioner. Further, the care provided to approximately two-thirds of the patients did not take comorbidities into account and insufficiently addressed more diffuse symptoms or problems. The review of the medication lists revealed that the majority of the medication lists contained inappropriate medications and that there were incongruity in medication listed in the primary and secondary care sector. Several barriers for providing high quality care were identified. These included relative short consultation times in general practice and outpatient clinics, lack of care coordinators, and lack of shared IT-system proving an overview of the treatment.

Conclusions: Our findings reveal quality of care deficiencies for people with multimorbidity. Suggestions for care improvement for people with multimorbidity includes formally assigned responsibility for care coordination, a change in the financial incentive structure towards a system rewarding high quality care and care focusing on prevention of disease exacerbation, as well as implementing shared medical record systems.
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http://dx.doi.org/10.1186/s12913-017-2724-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5694163PMC
November 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

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

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

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

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

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

Differential age-related changes in mitochondrial DNA repair activities in mouse brain regions.

Neurobiol Aging 2010 Jun 12;31(6):993-1002. Epub 2008 Aug 12.

Danish Center of Molecular Gerontology, University of Aarhus, Department of Molecular Biology, Aarhus C, Denmark.

Aging in the brain is characterized by increased susceptibility to neuronal loss and functional decline, and mitochondrial DNA (mtDNA) mutations are thought to play an important role in these processes. Due to the proximity of mtDNA to the main sites of mitochondrial free radical generation, oxidative stress is a major source of DNA mutations in mitochondria. The base excision repair (BER) pathway removes oxidative lesions from mtDNA, thereby constituting an important mechanism to avoid accumulation of mtDNA mutations. The complexity of the brain implies that exposure and defence against oxidative stress varies among brain regions and hence some regions may be particularly prone to accumulation of mtDNA damages. In the current study we investigated the efficiency of the BER pathway throughout the murine lifespan in mitochondria from cortex and hippocampus, regions that are central in mammalian cognition, and which are severely affected during aging and in neurodegenerative diseases. A regional specific regulation of mitochondrial DNA repair activities was observed with aging. In cortical mitochondria, DNA glycosylase activities peaked at middle-age followed by a significant drop at old age. However, only minor changes were observed in hippocampal mitochondria during the whole lifespan of the animals. Furthermore, DNA glycosylase activities were lower in hippocampal than in cortical mitochondria. Mitochondrial AP endonuclease activity increased in old animals in both brain regions. Our data suggest an important regional specific regulation of mitochondrial BER during aging.
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http://dx.doi.org/10.1016/j.neurobiolaging.2008.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858237PMC
June 2010