A continuous flow bioreactor system for high-throughput hyperpolarized metabolic flux analysis.

Hyperpolarized carbon-13 labeled compounds are increasingly being used in medical MR imaging (MRI) and MR imaging (MRI) and spectroscopy (MRS) research, due to its ability to monitor tissue and cell metabolism in real-time. Although radiological biomarkers are increasingly being considered as clinical indicators, biopsies are still considered the gold standard for a large variety of indications. Bioreactor systems can play an important role in biopsy examinations because of their ability to provide a physiochemical environment that is conducive for therapeutic response monitoring ex vivo.

Na,K-ATPase with Disrupted Na Binding Sites I and III Binds Na with Increased Affinity at Site II and Undergoes Na-Activated Phosphorylation with ATP.

Na,K-ATPase actively extrudes three cytoplasmic Na ions in exchange for two extracellular K ions for each ATP hydrolyzed. The atomic structure with bound Na identifies three Na sites, named I, II, and III. It has been proposed that site III is the first to be occupied and site II last, when Na binds from the cytoplasmic side.

On the track of the lipid transport pathway of the phospholipid flippase ATP8A2 - Mutation analysis of residues of the transmembrane segments M1, M2, M3 and M4.

P4-ATPases, also known as flippases, translocate specific lipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thereby generating an asymmetric lipid distribution essential for numerous cellular functions. A debated issue is which pathway within the protein the lipid substrate follows during the translocation. Here we present a comprehensive mutational screening of all amino acid residues in the transmembrane segments M1, M2, M3, and M4 of the flippase ATP8A2, thus allowing the functionally important residues in these transmembrane segments to be highlighted on a background of less important residues.

Temperature instability of a mutation at a multidomain junction in Na,K-ATPase isoform ATP1A3 (p.Arg756His) produces a fever-induced neurological syndrome.

ATP1A3 encodes the α3 isoform of Na,K-ATPase. In the brain, it is expressed only in neurons. Human ATP1A3 mutations produce a wide spectrum of phenotypes, but particular syndromes are associated with unique substitutions.

Electrogenic reaction step and phospholipid translocation pathway of the mammalian P4-ATPase ATP8A2.

ATP8A2 is a mammalian P4-ATPase (flippase) that translocates the negatively charged lipid substrate phosphatidylserine from the exoplasmic leaflet to the cytoplasmic leaflet of cellular membranes. Using an electrophysiological method based on solid supported membranes, we investigated the electrogenicity of specific reaction steps of ATP8A2 and explored a potential phospholipid translocation pathway involving residues with positively charged side chains. Changes to the current signals caused by mutations show that the main electrogenic event occurs in connection with the release of the bound phosphatidylserine to the cytoplasmic leaflet and support the hypothesis that the phospholipid interacts with specific lysine and arginine residues near the cytoplasmic border of the lipid bilayer during the translocation and reorientation required for insertion into the cytoplasmic leaflet.

Role of a conserved ion-binding site tyrosine in ion selectivity of the Na+/K+ pump.

The essential transmembrane Na+ and K+ gradients in animal cells are established by the Na+/K+ pump, a P-type ATPase that exports three Na+ and imports two K+ per ATP hydrolyzed. The mechanism by which the Na+/K+ pump distinguishes between Na+ and K+ at the two membrane sides is poorly understood. Crystal structures identify two sites (sites I and II) that bind Na+ or K+ and a third (site III) specific for Na+.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

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

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.

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

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 .

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.

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

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.

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

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.

The rapid-onset dystonia parkinsonism mutation D923N of the Na+, K+-ATPase alpha3 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 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(+).

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 E(1)-E(2) conformational changes, have been applied.

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.