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.
Electroencephalogram (EEG) interpretation plays a critical role in the clinical assessment of neurological conditions, most notably epilepsy. However, EEG recordings are typically analyzed manually ...by highly specialized and heavily trained personnel. Moreover, the low rate of capturing abnormal events during the procedure makes interpretation time-consuming, resource-hungry, and overall an expensive process. Automatic detection offers the potential to improve the quality of patient care by shortening the time to diagnosis, managing big data and optimizing the allocation of human resources towards precision medicine. Here, we present
, a novel unsupervised machine-learning method comprised of the interplay between an autoencoder network, a hidden Markov model (HMM), and a generative component: after dividing the signal into overlapping frames and performing a fast Fourier transform,
trains an autoencoder neural network for dimensionality reduction and compact representation of different frequency patterns for each frame. Next, we processed the temporal patterns using a HMM, while a third and generative component hypothesized and characterized the different phases that were then fed back to the HMM.
then automatically generates labels that the physician can interpret as pathological and non-pathological phases, thus effectively reducing the search space for trained personnel. We evaluated
's predictive performance on 686 recordings, encompassing more than 980 h from the publicly available Physionet database. Compared to manual annotations,
identified 197 of 198 epileptic events (99.45%), and is, as such, a highly sensitive method, which is a prerequisite for clinical use.
The sodium pump, Na,K‐ATPase, is an integral plasma membrane protein, expressed in all eukaryotic cells. Na,K‐ATPase transforms chemical energy from ATP into a gradient of Na+ and K+ over the plasma ...membrane by actively exporting three Na+ ions and importing two K+ ions for each hydrolyzed ATP. It is responsible for maintenance of the transmembrane Na+ gradient and is the major determinant of the membrane potential. It provides the driving force for all Na+‐coupled transport processes, thereby controlling essential functions in the cell. Na,K‐ATPase is formed by three subunits alpha/beta/FXYD, where alpha is the catalytic ion‐transporting subunit, beta is a regulatory subunit and FXYD is accessory.
Self‐interaction and oligomerization of the Na,K‐ATPase alpha/beta heterodimer in cell membranes has been proposed and discussed for a long time but is still an open question.
Here we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET‐FCS, in order to detect oligomers of Na,K‐ATPase. Compared to conventional cross‐correlation FCCS, FRET‐FCS is one to two orders of magnitude more sensitive when detecting oligomers. Moreover, FRET‐FCS is inherently insensitive to unbalanced labeling, which is a great advantage during live cell measurements.
We hypothesized that Na,K‐ATPase can exist in a higher order oligomeric state and demonstrate the use of FRET‐FCS to test this hypothesis
We have introduced fluorescent labels by using expression of non‐canonical amino acid modified beta subunits. The FRET pair Alexa488 and Alexa647 was directly conjugated to the beta subunits using selective click chemistry. Conventional FCS measurements of labeled cells revealed the absolute density of labeled and unlabeled Na,K‐ATPase. With FRET‐FCS we could observe FRET signals and FCS curves demonstrating the existence of oligomers. Positive controls for the FRET‐FCS measurements were constructed by labeling alpha subunits with Alexa488 and beta subunits with Alexa647.
Furthermore, we performed Monte Carlo simulations of Na,K‐ATPase, as monomer and as oligomer of increasing order, together with its ligands in a picket and fence diffusion model of the plasma membrane. The simulations suggest that oligomerization can have an impact on the net efficiency of the Na,K‐ATPase measured as ATP turnover.
In conclusion we find that Na,K‐ATPase can be found in the plasma membrane as oligomers. Further we discuss the consequences of oligomerization and propose that it can have a regulatory effect for the Na,K‐ATPase net efficiency.
Most neurons co-express two catalytic isoforms of Na,K-ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific ...role of this isoform is not completely understood. Here, we used electrophysiological and Na+ imaging techniques to study the role of α3 in central nervous system neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside, ouabain, resulted in a modest increase in intracellular Na+ concentration (Na+i) accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in Na+i, similar to what could be expected following suprathreshold neuronal activity, selective inhibition of α3 almost completely abolished the capacity to restore Na+i in soma and dendrite. Recordings of Na,K-ATPase specific current supported the notion that when Na+i is elevated in the neuron, α3 is the predominant isoform responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A consensus site, which is structurally associated with an Na+ binding site. Following activation of protein kinase A, both the α3-dependent current and restoration of dendritic Na+i were significantly attenuated, indicating that α3 is a target for phosphorylation and may participate in short term regulation of neuronal function.
Background: Neurons express two Na,K-ATPase isoforms, the ubiquitous α1 and neuron-specific α3.
Results: α3 is important for control of membrane potential and is fully responsible for restoration of large Na+i increases.
Conclusion: α1 and α3 are required for basal neuronal function, but α3 controls restoration of Na+i following sustained discharge.
Significance: Conditions associated with defect α3 function are likely aggravated by suprathreshold neuronal activity.
ABSTRACTThe 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. FASEB J. 33, 10193–10206 (2019). www.fasebj.org
Exposure of intact cells to selective inhibitors of Na+/K+-ATPase such as ouabain activates several growth-related cell signaling pathways. It has been suggested that the initial event of these ...pathways is the binding of ouabain to a preexisting complex of Src with Na+/K+-ATPase of the plasma membrane. The aim of this work was to evaluate the role of Src in the ouabain-induced activation of phosphatidylinositide 3-kinase 1A (PI3K1A) and its downstream consequences. When fibroblasts devoid of Src (SYF cells) and controls (Src++ cells) were exposed to ouabain, PI3K1A, Akt, and proliferative growth were similarly stimulated in both cell lines. Ouabain-induced activation of Akt was not prevented by the Src inhibitor PP2. In contrast, ERK1/2 were not activated by ouabain in SYF cells but were stimulated in Src++ cells; this was prevented by PP2. In isolated adult mouse cardiac myocytes, where ouabain induces hypertrophic growth, PP2 also did not prevent ouabain-induced activation of Akt and the resulting hypertrophy. Ouabain-induced increases in the levels of co-immunoprecipitation of the α-subunit of Na+/K+-ATPase with the p85 subunit of PI3K1A were noted in SYF cells, Src++ cells, and adult cardiac myocytes. In conjunction with previous findings, the results presented here indicate that (a) if there is a preformed complex of Src and Na+/K+-ATPase, it is irrelevant to ouabain-induced activation of the PI3K1A/Akt pathway through Na+/K+-ATPase and (b) a more likely, but not established, mechanism of linkage of Na+/K+-ATPase to PI3K1A is the ouabain-induced interaction of a proline-rich domain of the α-subunit of Na+/K+-ATPase with the SH3 domain of the p85 subunit of PI3K1A.
The sodium potassium pump, Na,K-ATPase (NKA), is an integral plasma membrane protein, expressed in all eukaryotic cells. It is responsible for maintaining the transmembrane Na+ gradient and is the ...major determinant of the membrane potential. Self-interaction and oligomerization of NKA in cell membranes has been proposed and discussed but is still an open question. Here, we have used a combination of FRET and Fluorescence Correlation Spectroscopy, FRET-FCS, to analyze NKA in the plasma membrane of living cells. Click chemistry was used to conjugate the fluorescent labels Alexa 488 and Alexa 647 to non-canonical amino acids introduced in the NKA α1 and β1 subunits.
We demonstrate that FRET-FCS can detect an order of magnitude lower concentration of green-red labeled protein pairs in a single-labeled red and green background than what is possible with cross-correlation (FCCS). We show that a significant fraction of NKA is expressed as a dimer in the plasma membrane. We also introduce a method to estimate not only the number of single and double labeled NKA, but the number of unlabeled, endogenous NKA and estimate the density of endogenous NKA at the plasma membrane to 1400 ± 800 enzymes/μm2.
Display omitted
•FRET-FCS is more sensitive than FCCS for detection of rare dual labeled molecules.•Na,K-ATPase can be found as both monomers and dimers in the plasma membrane.•The density of Na,K-ATPase in the HEK293T cell membrane is 1400 ± 800 enzymes/μm2.
NMDA receptors play a crucial role in regulating synaptic plasticity and memory. Activation of NMDA receptors changes intracellular concentrations of Na
+
and K
+
, which are subsequently restored by ...Na/K-ATPase. We used immunochemical and biochemical methods to elucidate the potential mechanisms of interaction between these two proteins. We observed that NMDA receptor and Na/K-ATPase interact with each other and this interaction was shown for both isoforms of α subunit (α1 and α3) of Na/K-ATPase expressed in neurons. Using Western blotting, we showed that long-term exposure of the primary culture of cerebellar neurons to nanomolar concentrations of ouabain (a cardiotonic steroid, a specific ligand of Na/K-ATPase) leads to a decrease in the levels of NMDA receptors which is likely mediated by the α3 subunit of Na/K-ATPase. We also observed a decrease in enzymatic activity of the α1 subunit of Na/K-ATPase caused by NMDA receptor activation. This effect is mediated by an increase in intracellular Ca
2+
. Thus, Na/K-ATPase and NMDA receptor can interact functionally by forming a macromolecular complex which can be important for restoring ionic balance after neuronal excitation. Furthermore, this interaction suggests that NMDA receptor function can be regulated by endogenous cardiotonic steroids which recently have been found in cerebrospinal fluid or by pharmacological drugs affecting Na/K-ATPase function.
Chlamydia are ancient intracellular pathogens with reduced, though strikingly conserved genome. Despite their parasitic lifestyle and isolated intracellular environment, these bacteria managed to ...avoid accumulation of deleterious mutations leading to subsequent genome degradation characteristic for many parasitic bacteria.
We report pan-genomic analysis of sixteen species from genus Chlamydia including identification and functional annotation of orthologous genes, and characterization of gene gains, losses, and rearrangements. We demonstrate the overall genome stability of these bacteria as indicated by a large fraction of common genes with conserved genomic locations. On the other hand, extreme evolvability is confined to several paralogous gene families such as polymorphic membrane proteins and phospholipase D, and likely is caused by the pressure from the host immune system.
This combination of a large, conserved core genome and a small, evolvable periphery likely reflect the balance between the selective pressure towards genome reduction and the need to adapt to escape from the host immunity.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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 α1 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 α1 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 α1 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 α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 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.
PDF
