Rikke Holm

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

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.

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

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 E1-E2 conformational changes, have been applied. The results pinpoint the two terminal tyrosines, Tyr1017 and Tyr1018, as well as putative interaction partners, Arg935 in the loop between transmembrane segments M8 and M9 and Lys768 in transmembrane segment M5, as crucial to Na+ activation of phosphorylation of E1, a partial reaction reflecting Na+ interaction on the cytoplasmic side of the membrane. Tyr1017, Tyr1018, and Arg935 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 E1P to E2P backwards toward E1P and inhibit Na+-ATPase activity in mutants. Lys768 is not important for Na+ binding from the external side of the membrane but is involved in stabilization of the E2 form. These data demonstrate that the C terminus controls Na+ affinity on both sides of the membrane and suggest that Arg935 constitutes an important link between the C terminus and the third Na+ site, involving an arginine-π stacking interaction between Arg935 and the C-terminal tyrosines. Lys768 may interact preferentially with the C terminus in E1 and E1P forms and with the loop between transmembrane segments M6 and M7 in E2 and E2P forms.

The Rapid-onset Dystonia Parkinsonism Mutation D923N of the Na+,K+-ATPase α3 Isoform Disrupts Na+ Interaction at the Third Na+ Site

Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific α3-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 ∼200-fold reduction of Na+ affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E1 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 E1P to E2P conformational transition backward. However, no impairment of K+ binding was observed for D923N, leading to the conclusion that Asp923 is specifically associated with the third Na+ site that is selective toward Na+. The crystal structure of the Na+,K+-ATPase in E2 form shows that Asp923 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 E1 form of Na+,K+-ATPase based on the Ca2+-ATPase crystal structure is consistent with the hypothesis that Asp923 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.

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

Background: Na+,K+-ATPase mutations extending the C terminus cause neurological disease. Results: C-terminal extension reduces Na+ affinity. Analysis of several mutants establishes a relationship between change in Na+ affinity and change of intracellular Na+ and K+ concentrations. Conclusion: The Na+ affinity of the Na+,K+-ATPase is a major in vivo determinant of the intracellular Na+ concentration. Significance: Insight in pathophysiology and regulation of the Na+,K+-ATPase is obtained. 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.

Inhibition of Phosphorylation of Na+,K+-ATPase by Mutations Causing Familial Hemiplegic Migraine

Background: Familial hemiplegic migraine type II (FHM2) is caused by mutations in the Na+,K+-ATPase α2-isoform. Results: Several FHM2 mutations inhibit phosphorylation or dephosphorylation. Conclusion: These mutations cause FHM2 by local and long range effects on the catalytic site and not by reducing the affinity for external K+. Significance: Insights into the pathophysiological mechanism of FHM2 and the molecular mechanism of the Na+,K+-ATPase have been obtained. 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 E2P 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 E1P to E2P transition.

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

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.

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

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+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.

Importance of a potential protein kinase A phosphorylation site of Na+,K+-ATPase and its interaction network for Na+ binding

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.

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

Significance This study explores the structural basis for the difference between the closely related Na+,K+-ATPase and H+,K+-ATPase with respect to ion transport stoichiometry, electrogenicity, and cation selectivity, which has long been an enigma. A cysteine in the membrane domain of Na+,K+-ATPase is substituted by arginine in H+,K+-ATPase and has been found to be replaced by phenylalanine in a patient with alternating hemiplegia of childhood. These substitutions affect the functioning of the Na+-specific site of Na+,K+-ATPase profoundly. The positively charged side chain of the arginine present in H+,K+-ATPase serves as an internal cation that replaces bound Na+, thus explaining how H+,K+-ATPase avoids binding and transporting Na+ at this site, thereby performing electroneutral transport, despite the general structural and functional resemblance to the electrogenic Na+,K+-ATPase. 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.

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

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.