Mutations in the gene encoding the alpha3 Na+/K+-ATPase isoform (ATP1A3) lead to movement disorders that manifest with dystonia, a common neurological symptom with many different origins, but for which the underlying molecular mechanisms remain poorly understood. We have generated an ATP1A3 mutant mouse that displays motor impairments and a hyperexcitable motor phenotype compatible with dystonia. We show that neurons harbouring this mutation are compromised in their ability to extrude raised levels of intracellular sodium, highlighting a profound deficit in neuronal sodium homeostasis. We show that the spinal motor network in ATP1A3 mutant mice has a reduced responsiveness to activity-dependent rises in intracellular sodium and that this is accompanied by loss of the Na+/K+-ATPase-mediated afterhyperpolarization in motor neurons. Taken together, our data support that the alpha3 Na+/K+-ATPase is important for cellular and spinal motor network homeostasis. These insights suggest that it may be useful to consider ways to compensate for this loss of a critical afterhyperpolarization-dependent control of neuronal excitability when developing future therapies for dystonia.

Activation of the apoptotic pathway is a major cause of progressive loss of function in chronic diseases such as neurodegenerative and diabetic kidney diseases. There is an unmet need for an anti-apoptotic drug that acts in the early stage of the apoptotic process. The multifunctional protein Na + ,K + -ATPase has, in addition to its role as a transporter, a signaling function that is activated by its ligand, the cardiotonic steroid ouabain. Several lines of evidence suggest that sub-saturating concentrations of ouabain protect against apoptosis of renal epithelial cells, a common complication and major cause of death in diabetic patients. Here, we induced apoptosis in primary rat renal epithelial cells by exposing them to an elevated glucose concentration (20 mM) and visualized the early steps in the apoptotic process using super-resolution microscopy. Treatment with 10 nM ouabain interfered with the onset of the apoptotic process by inhibiting the activation of the BH3-only protein Bad and its translocation to mitochondria. This occurred before the pro-apoptotic protein Bax had been recruited to mitochondria. Two ouabain regulated and Akt activating Ca 2+ /calmodulin-dependent kinases were found to play an essential role in the ouabain anti-apoptotic effect. Our results set the stage for further exploration of ouabain as an anti-apoptotic drug in diabetic kidney disease as well as in other chronic diseases associated with excessive apoptosis.

Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na + /K + ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.

The ion pump Na+,K+-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na+,K+-ATPase catalytic alpha 1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg2+. We found that membrane expression of the mutant alpha 1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na+,K+-ATPase transport demonstrated massive water inflow into mutant alpha 1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant alpha 1 detected a nonspecific cation current. Finally, neurons expressing mutant alpha 1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na+,K+-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.

Na,K-ATPase is a ubiquitous multifunctional protein that acts both as an ion pump and as a signal transducer. The signaling function is activated by ouabain in non-toxic concentrations. In epithelial cells the ouabain-bound Na,K-ATPase connects with the inositol 1,4,5-trisphosphate receptor via a short linear motif to activate low frequency Ca2+ oscillations. Within a couple of minutes this ouabain mediated signal has resulted in phosphorylation or dephosphorylation of 2580 phospho-sites. Proteins that control cell proliferation and cell adhesion and calmodulin regulated proteins are enriched among the ouabain phosphor-regulated proteins. The inositol 1,4,5-trisphosphate receptor and the stromal interaction molecule, which are both essential for the initiation of Ca2+ oscillations, belong to the ouabain phosphor-regulated proteins. Downstream effects of the ouabain-evoked Ca2+ signal in epithelial cells include interference with the intrinsic mitochondrial apoptotic process and stimulation of embryonic growth processes. The dual function of Na,K-ATPase as an ion pump and a signal transducer is now well established and evaluation of the physiological and pathophysiological consequences of this universal signal emerges as an urgent topic for future studies.

Given its critical role in establishing ionic gradients in neurons and other excitable cells, functional disruption of the Na/K‐ATPase is expected to substantially compromise neurological homeostasis. However, to date only a few examples of spontaneous Na/K‐ATPase mutations have been identified in human patients. Here we describe a de novo Trp‐Arg mutation in the ATP1A1 gene, encoding the Na/K‐ATPase catalytic α1 subunit, in an infant who died at 10 months from therapy‐resistant epilepsy. Located in a conserved transmembrane helical region of the α1 subunit, the mutation was associated primarily with neurological symptoms and a low capacity to retain magnesium. Confocal imaging of the mutant Na/K‐ATPase α1 subunit expressed in rat hippocampal neurons revealed decreased but detectable membrane expression. Expressed in Xenopus oocytes, the mutant Na/K‐ATPase catalytic α1 subunit was associated with nonselective, ouabain‐resistant leak currents, consistent with a sizable, continuous hydration defect in the transmembrane domain. Indeed, molecular dynamics simulations in both POPC and mixed‐lipid bilayers showed accumulation of water at the protein periphery, particularly in the vicinity of crystallographic Na‐binding sites. These results provide evidence, from clinical diagnosis to atomistic simulation, for a mechanism of ion‐gradient disruption that could account for leak currents now observed in multiple pathological Na/K‐ATPase variants. Our finding of a pathological interaction between the mutated ion transporter and the membrane lipids has implications for development of new treatment strategy in many cases of genetic epilepsy.Support or Funding InformationSwedish Research Council to AA and to HB

The N-methyl-D-aspartate (NMDA) receptor plays an essential role in glutamatergic transmission and synaptic plasticity and researchers are seeking for different modulators of NMDA receptor function. One possible mechanism for its regulation could be through adjacent membrane proteins. NMDA receptors coprecipitate with Na,K-ATPase, indicating a potential interaction of these two proteins. Ouabain, a mammalian cardiotonic steroid that specifically binds to Na,K-ATPase and affects its conformation, can protect from some toxic effects of NMDA receptor activation. Here we have examined whether NMDA receptor activity and downstream effects can be modulated by physiological ouabain concentrations. The spatial colocalization between NMDA receptors and the Na,K-ATPase catalytic subunits on dendrites of cultured rat hippocampal neurons was analyzed with super-resolution dSTORM microscopy. The functional interaction was analyzed with calcium imaging of single hippocampal neurons exposed to 10 μM NMDA in presence and absence of ouabain and by determination of the ouabain effect on NMDA receptor–dependent long-term potentiation. We show that NMDA receptors and the Na,K-ATPase catalytic subunits alpha1 and alpha3 exist in same protein complex and that ouabain in nanomolar concentration consistently reduces the calcium response to NMDA. Downregulation of the NMDA response is not associated with internalization of the receptor or with alterations in its state of Src phosphorylation. Ouabain in nanomolar concentration elicits a long-term potentiation response. Our findings suggest that ouabain binding to a fraction of Na,K-ATPase molecules that cluster with the NMDA receptors will, via a conformational effect on the NMDA receptors, cause moderate but consistent reduction of NMDA receptor response at synaptic activation.

The ion pump Na+, K+-ATPase (NKA) is a receptor for the cardiotonic steroid ouabain. Subsaturating concentration of ouabain triggers intracellular calcium oscillations, stimulates cell proliferation and adhesion, and protects from apoptosis. However, it is controversial whether ouabain-bound NKA is considered a signal transducer. To address this question, we performed a global analysis of protein phosphorylation in COS-7 cells, identifying 2580 regulated phosphorylation events on 1242 proteins upon 10- and 20-min treatment with ouabain. Regulated phosphorylated proteins include the inositol triphosphate receptor and stromal interaction molecule, which are essential for initiating calcium oscillations. Hierarchical clustering revealed that ouabain triggers a structured phosphorylation response that occurs in a well-defined, time-dependent manner and affects specific cellular processes, including cell proliferation and cell-cell junctions. We additionally identify regulation of the phosphorylation of several calcium and calmodulin-dependent protein kinases (CAMKs), including 2 sites of CAMK type II-γ (CAMK2G), a protein known to regulate apoptosis. To verify the significance of this result, CAMK2G was knocked down in primary kidney cells. CAMK2G knockdown impaired ouabain-dependent protection from apoptosis upon treatment with high glucose or serum deprivation. In conclusion, we establish NKA as the coordinator of a broad, tightly regulated phosphorylation response in cells and define CAMK2G as a downstream effector of NKA.-Panizza, E., Zhang, L., Fontana, J. M., Hamada, K., Svensson, D., Akkuratov, E. E., Scott, L., Mikoshiba, K., Brismar, H., Lehtiö, J., Aperia, A. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+, K+-ATPase control of cell adhesion, proliferation, and survival.

It is generally believed that cells that are unable to downregulate glucose transport are particularly vulnerable to hyperglycemia. Yet, little is known about the relation between expression of glucose transporters and acute toxic effects of high glucose exposure. In the present ex vivo study of rat renal cells, we compared the apoptotic response to a moderate increase in glucose concentration. We studied cell types that commonly are targeted in diabetic kidney disease (DKD): proximal tubule cells, which express Na+-dependent glucose transporter (SGLT)2, mesangial cells, which express SGLT1, and podocytes, which lack SGLT and take up glucose via insulin-dependent glucose transporter 4. Proximal tubule cells and mesangial cells responded within 4–8 h of exposure to 15 mM glucose with translocation of the apoptotic protein Bax to mitochondria and an increased apoptotic index. SGLT downregulation and exposure to SGLT inhibitors abolished the apoptotic response. The onset of overt DKD generally coincides with the onset of albuminuria. Albumin had an additive effect on the apoptotic response. Ouabain, which interferes with the apoptotic onset, rescued from the apoptotic response. Insulin-supplemented podocytes remained resistant to 15 and 30 mM glucose for at least 24 h. Our study points to a previously unappreciated role of SGLT-dependent glucose uptake as a risk factor for diabetic complications and highlights the importance of therapeutic approaches that specifically target the different cell types in DKD.

Objectives Diabetic nephropathy (DN) is the most common cause of end‐stage renal failure and affects around 40% of all diabetic patients. This project aims to investigate the loss of proximal tubular cells (PTCs) in DN which are epithelial cells of the nephron leading away from the Bowman's capsule. Hyperglycemia induced damage to PTCs is attenuated by the Na/K‐ATPase ligand ouabain. Our second objective was thus to investigated the underlying mechanisms of ouabain in vitro and the function of Na/K‐ATPase as a signaling transducer. Methods PTCs were isolated from mouse and rat kidney cortex and cultured for up to a week. COS‐7 kidney cells were used for some experiments. Protein expression and phosphorylation was studied by Western blotting and proteomics/phosphoproteomics. Gene ontology analysis was performed to find distinct cellular processes regulated by ouabain in the phopshoproteomic analysis. Seahorse was used for metabolic studies. Gene expression was analyzed by qPCR and apoptosis assessed by the TUNEL assay. Key results High glucose (HG, 15 mM) triggers apoptosis in PTC due to their expression of sodium dependent glucose transporter 2 (SGLT2), SGLT2 siRNA abolishes HG induced apoptosis. Apoptosis does not appear to coincide with a switch in glucose metabolism. Low concentrations of ouabain (10 nM) completely inhibits the apoptosis. Phosphoproteomic analysis revealed 2580 ouabain‐regulated phosphorylation sites, many of which were associated with cell adhesion and proliferation. Out of these, CaMK2γ were shown to be involved in the anti‐apoptotic effect of ouabain, siRNA silencing of CaMK2γ eliminates the protective effect of ouabain. Conclusions Loss of tubular cells in DN might be driven by hyperglycemia due to the PTC expression of SGLT2. Low concentrations of ouabain protects from the detrimental hyperglycemic effects through an anti‐apoptotic mechanism involving CaMK2γ. Support or Funding Information The study was funded by the Swedish Research Council and Erling‐Persson Family Foundation. D.S. and N.M. is supported by a Novo Nordisk postdoctoral fellowship run in partnership with Karolinska Institutet. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Neuronal activity leads to an influx of Na+ that needs to be rapidly cleared. The sodium-potassium ATPase (Na,K-ATPase) exports three Na+ ions and imports two K+ ions at the expense of one ATP molecule. Na,K-ATPase turnover accounts for the majority of energy used by the brain. To prevent an energy crisis, the energy expense for Na+ clearance must provide an optimal effect. Here we report that in rat primary hippocampal neurons, the clearance of Na+ ions is more efficient if Na,K-ATPase is laterally mobile in the membrane than if it is clustered. Using fluorescence recovery after photobleaching and single particle tracking analysis, we show that the ubiquitous α1 and the neuron-specific α3 catalytic subunits as well as the supportive β1 subunit of Na,K-ATPase are highly mobile in the plasma membrane. We show that cross-linking of the β1 subunit with polyclonal antibodies or exposure to Modulator of Na,K-ATPase (MONaKA), a secreted protein which binds to the extracellular domain of the β subunit, clusters the α3 subunit in the membrane and restricts its mobility. We demonstrate that clustering, caused by cross-linking or by exposure to MONaKA, reduces the efficiency in restoring intracellular Na+. These results demonstrate that extracellular interactions with Na,K-ATPase regulate the Na+ extrusion efficiency with consequences for neuronal energy balance.

The central role of calcium signaling during development of early vertebrates is well documented, but little is known about its role in mammalian embryogenesis. We have used immunofluorescence and time-lapse calcium imaging of cultured explanted embryonic rat kidneys to study the role of calcium signaling for branching morphogenesis. In mesenchymal cells, we recorded spontaneous calcium activity that was characterized by irregular calcium transients. The calcium signals were dependent on release of calcium from intracellular stores in the endoplasmic reticulum. Down-regulation of the calcium activity, both by blocking the sarco-endoplasmic reticulum Ca2+-ATPase and by chelating cytosolic calcium, resulted in retardation of branching morphogenesis and a reduced formation of primitive nephrons but had no effect on cell proliferation. We propose that spontaneous calcium activity contributes with a stochastic factor to the self-organizing process that controls branching morphogenesis, a major determinant of the ultimate number of nephrons in the kidney.Fontana, J. M., Khodus, G. R., Unnersjo-Jess, D., Blom, H., Aperia, A., Brismar, H. Spontaneous calcium activity in metanephric mesenchymal cells regulates branching morphogenesis in the embryonic kidney.

Background Blockers of angiotensin II type 1 receptor (AT 1 R) and the voltage gated calcium channel 1.2 (Ca V 1.2) are commonly used for treatment of hypertension. Yet there is little information about the effect of physiological concentrations of angiotensin II (AngII) on AT 1 R signaling and whether there is a reciprocal regulation of AT 1 R signaling by Ca V 1.2. Methods To elucidate these questions, we have studied the Ca 2+ signaling response to physiological and pharmacological AngII doses in HEK293a cells, vascular smooth muscle cells and cardiomyocytes using a Ca 2+ sensitive dye as the principal sensor. Intra-cellular calcium recordings were performed in presence and absence of Ca V 1.2 blockers. Semi-quantitative imaging methods were used to assess the plasma membrane expression of AT 1 R and G-protein activation. Results Repeated exposure to pharmacological (100 nM) concentrations of AngII caused, as expected, a down-regulation of the Ca 2+ response. In contrast, repeated exposure to physiological (1 nM) AngII concentration resulted in an enhancement of the Ca 2+ response. The up-regulation of the Ca 2+ response to repeated 1 nM AngII doses and the down-regulation of the Ca 2+ response to repeated 100 nM Angll doses were not accompanied by a parallel change of the AT 1 R plasma membrane expression. The Ca 2+ response to 1 nM of AngII was amplified in the presence of therapeutic concentrations of the Ca V 1.2 blockers, nifedipine and verapamil, in vascular smooth muscle cells, cardiomyocytes and HEK293a cells. Amplification of the AT 1 R response was also observed following inhibition of the calcium permeable transient receptor potential cation channels, suggesting that the activity of AT 1 R is sensitive to calcium influx. Conclusions Our findings have implications for the understanding of hyperactivity of the angiotensin system and for use of Ca 2+ channel blockers as mono-therapy in hypertension.

The N‐methyl‐d‐aspartate (NMDA) receptor plays an essential role for glutamatergic transmission and synaptic plasticity. Calcium influx via the NMDA receptor (NMDAR) activation modulates both long‐term potentiation (LTP) and long‐term depression (LTD). Hyperactivity of the NMDAR can trigger events leading to calcium mediated neurotoxicity, a common cause of brain damage. Recent studies have suggested that the Na+,K+‐ATPase (NKA) may interact with NMDAR and that ouabain, a highly specific NKA ligand, has a neuro‐protective effect. Here we have tested whether subsaturating concentrations of ouabain may attenuate the calcium response from an activated NMDAR. Single hippocampal neurons derived from E18.5 rats and cultured for 3 weeks were exposed to 10μM NMDA with or without 10nM ouabain, and the dendritic calcium response was recorded. The calcium influx was immediately and transiently attenuated with approximately 40% when ouabain was co‐administered. The ouabain effect was not due to NMDAR internalization, changes in membrane potential or alterations in Src family phosphorylation. Studies on HEK cells expressing NMDAR subunits, the ubiquitous NKA catalytic alpha1 subunit and the neuron specific NKA alpha3 subunit, confirmed that ouabain attenuates the calcium response from an activated NMDAR and indicated that ouabain attenuation of NMDAR calcium response was mediated via the alpha3 and not the NKA alpha1 subunit . Since ouabain locks NKA alpha subunits in one of its two major conformations, we speculated that ouabain bound NKA alpha3 can act as an allosteric modifier of NMDAR. Using dual‐color direct stochastic optical reconstruction microscopy (dSTORM) we showed a spatial correlation between the NMDARs and the NKA alpha1 and alpha3 subunits. The distance between NKA alpha3 and the NR2 subunits of the NMDAR was less than 50 nm for 50% of the NKA alpha3 and less than 70 nm for 50% of the NKA alpha1 subunits. In summary, our results demonstrate a novel mechanism for modulation of NMDAR signaling and offer an explanation for the neuro‐protective effect of ouabain reported by other investigators.

The role of the Bcl-family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods in studies of isolated mitochondria and transfected cell lines. T ...

“No research without trained researchers” has become the mantra of the EU-funded Innovative Medicines Initiative (IMI) education and training projects. However, it is often hard to determine the type of training required at different stages of a scientist’s career. The situation is further complicated by the constantly changing environment, e.g. the growth of disruptive technologies, societal expectations of biomedical sciences, the greater need for multi-disciplinary collaborations, and conservative or changing regulatory requirements. This article summarises the experience from a series of five EMTRAIN Public Private Partnership PhD workshops that included both scientific and transferrable skill training. This is followed by an example of a recently developed training programme, including a competency profile, for translational research and medicines development; the C-COMEND teaching programme. The emphasis is on competencies as a new currency for continuing professional development. Finally, this paper describes what we consider to be the next steps required by the scientific community to address solutions to the current training challenges so that society can benefit from the innovations that only science can provide.

The role of the Bcl‐family proteins in the mitochondrial apoptotic process is well described with biochemical and molecular methods as well as with studies of isolated mitochondria and transfected cell lines. There is yet no proof of principle that this description of apoptosis conforms with the in vivo process in primary mammalian cells. Here we have used Stimulated Emission Depletion (STED) microscopy to study the apoptotic process in immune‐stained rat renal epithelia cells exposed to 20 mM glucose (HG) and to study its rescue by ouabain. To assess distance between Bcl proteins, we used the nearest‐neighbor algorithm. The anti‐apoptotic protein Bcl‐xl was predominantly expressed on mitochondria in control cells, and remained so throughout the process, although its abundance decreased. After 2h HG the apoptosis‐inducing protein BAD had translocated from the cytoplasm to the mitochondria where it clustered with Bcl‐xL. This occurred before an increase in reactive oxygen species and was dependent on activation of the PI3K –AKT pathway. According to current concepts, Bcl‐xl interacts with the apoptotic protein Bax on the mitochondria under control conditions to translocate Bax back to the cytosol. We found that Bax started to accumulate on the mitochondria after 4h HG and, surprisingly, that the interaction between Bcl‐xL and Bax became more pronounced during the course of the apoptotic process. After 6h HG Bax also interacted with the non‐specific ion transporter VDAC; an interaction described to lead to penetration of the inner mitochondrial membrane and mark the point of no return. Our group has previously identified an anti‐apoptotic signaling pathway that is triggered by ouabain binding to Na,K‐ATPase and activates PI3K and AKT. Here we show that 10 nM ouabain rescues from apoptosis by counteracting the AKT dependent inhibition of BAD activation. This study reveals new information about interaction between Bcl family of proteins during the apoptotic process and about mechanism and rescue from kidney cell death in poorly controlled diabetes.

Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling.

The Na(+)-K(+)-ATPase (NKA) differs from most other ion transporters, not only in its capacity to maintain a steep electrochemical gradient across the plasma membrane, but also as a receptor for a family of cardiotonic steroids, to which ouabain belongs. Studies from many groups, performed during the last 15 years, have demonstrated that ouabain, a member of the cardiotonic steroid family, can activate a network of signaling molecules, and that NKA will also serve as a signal transducer that can provide a feedback loop between NKA and the mitochondria. This brief review summarizes the current knowledge and controversies with regard to the understanding of NKA signaling.