Background: Neurons express two Na,K-ATPase isoforms, the ubiquitous ␣1 and neuron-specific ␣3. Results: ␣3 is important for control of membrane potential and is fully responsible for restoration of large [Na ϩ ] i increases.

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.

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.

Background 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. Results 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. Conclusions 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.

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.

Studies in experimental systems have identified a multitude of mutational mechanisms including DNA replication infidelity and DNA damage followed by inefficient repair or replicative bypass. However, the relative contributions of these mechanisms to human germline mutation remain unknown. Here, we show that error-prone damage bypass on the lagging strand plays a major role in human mutagenesis. Transcription-coupled DNA repair removes lesions on the transcribed strand; lesions on the non-transcribed strand are preferentially converted into mutations. In human polymorphism we detect a striking similarity between mutation types predominant on non-transcribed strand and on the strand lagging during replication. Moreover, damage-induced mutations in cancers accumulate asymmetrically with respect to the direction of replication, suggesting that DNA lesions are resolved asymmetrically. We experimentally demonstrate that replication delay greatly attenuates the mutagenic effect of UV-irradiation confirming that replication converts DNA damage into mutations. We estimate that at least 10% of human mutations arise due to DNA damage.

Ouabain is a cardiotonic steroid with a history of medical uses. It acts as a ligand to the ubiquitous ion pump Na+,K+‐ATPase which it inhibits at high concentrations. At lower concentrations it triggers calcium oscillations and activation of MAP kinases, stimulates cell proliferation as well as adhesion and acts anti‐apoptotic. In light of this, it has been proposed that the pump may have a dual function as a signaling transducer. To investigate this, we have analyzed the phosphoproteome of COS‐7 kidney cells after treatment with sub‐saturating levels (100 nM) of ouabain, which were high enough to induce Ca2+ oscillations but below those needed to modulate the ion pumping function of Na+,K+‐ATPase. Gene ontology analysis was performed to find distinct cellular processes regulated by ouabain after 10 and 20 min of treatment in the phopshoproteomic analysis. The analysis revealed 2580 ouabain‐regulated phosphorylation sites, many of which were associated with cell adhesion and proliferation. Two of the regulated phospho‐proteins were the inositol triphosphoate receptor (InsP3) and stromal interaction molecule (STIM), both of which are essential for ouabain‐induced calcium oscillations. We used siRNA and Western blotting for target confirmation. One confirmed target, calcium/calmodulin‐dependent protein kinase II gamma (CAMK2γ), were shown to be involved in the anti‐apoptotic effect of ouabain; siRNA silencing of CaMK2γ in primary renal cells eliminates the protective effect of ouabain against apoptosis induced by either serum starvation or glucose. In conclusion, our findings support a role for Na+,K+‐ATPase as a signal transducer that serves to protect cell and tissue integrity. Support or Funding Information This study was supported by the Swedish Research Council and Erling‐Persson Family Foundation. D.S. 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.

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