Search Results (40)

Drug-resistant epilepsy remains a therapeutic challenge, requiring new molecular targets beyond conventional antiepileptic drugs. Small-conductance calcium-activated potassium (SK) channels and Na/K-ATPase (NKA) contribute to afterhyperpolarization via distinct mechanisms, offering complementary ways to suppress hyperexcitability. We examined SK activation and NKA modulation in synchronized epileptiform activity in a primary culture of cortical neurons obtained from rat embryos. Epileptiform discharges were induced by magnesium-free solution and assessed by patch-clamp and calcium imaging. The SK2/3 activator CyPPA (10 µM) reduced epileptiform current (EC) amplitude and integral and decreased synchronized calcium transient (CT) frequency but gradually elevated basal calcium. In contrast, ouabain (1 nM), a selective modulator of high-affinity NKA isoforms, attenuated EC amplitude, strongly suppressed CTs, and showed persistent effects after washout, accompanied by asynchronous glial calcium activity. Co-application of CyPPA with ouabain abolished CyPPA-induced calcium elevation while maintaining suppression of neuronal synchrony. The broader SK/IK activator NS309 (10 µM) reduced CT frequency and basal calcium without affecting glia. Thus, SK activation and NKA signaling suppress epileptiform synchronization through distinct yet convergent pathways: SK channels via afterhyperpolarization and NKA via afterhyperpolarization and calcium-dependent signaling. Their combination enhances efficacy and prevents adverse calcium buildup, supporting SK–NKA co-targeting as a strategy against drug-resistant epilepsy. Full article

AMOG/β₂, the β₂ isoform of the sodium pump (Na⁺/K⁺-ATPase), functions as an adhesion molecule on glial cells, mediating critical neuron–astrocyte interactions during central nervous system (CNS) development. Despite its established role in glial adhesion, the neuronal receptor that partners with AMOG/β₂ remains unknown. This review examines the structural and functional properties of AMOG/β₂, including its capacity to form trans-dimers, both homophilic and potentially heterophilic—drawing comparisons with the β₁ subunit, a well-characterized adhesion molecule. By integrating computational modeling, in vitro data, and structural predictions, we explore how factors such as N-glycosylation and cis-membrane interactions influence β₂-mediated adhesion. We further consider candidate neuronal partners, including TSPAN31 and RTN4, and speculate on their potential roles in mediating heterophilic AMOG/β₂ interactions. Finally, we discuss the broader implications of AMOG/β₂ in neuron–glia communication, synaptic organization, neurodevelopment, and CNS disorders such as glioblastoma. Identifying the binding partner of AMOG/β₂ holds promise not only for understanding the molecular basis of CNS adhesion but also for uncovering novel mechanisms of neuroglial regulation in health and disease. Full article

Cardiac glycosides (CGs), historically used to treat heart failure and arrhythmias, bind to the α subunit of the Na⁺/K⁺-ATPase pump and inhibit its activity. Their anticancer and antiviral activities are of interest. The α subunit of the Na⁺/K⁺-ATPase pump has four isoforms (α1–4), each with unique tissue distribution and expression pattern; their contributions to antiviral activities have not been studied. We previously reported that CGs inhibit human CMV (HCMV) in vitro but not mouse CMV (MCMV). In addition to the low affinity of mouse α1 for CGs, we hypothesized that other isoforms contribute to the anti-CMV activities of CGs. We show here that infection with HCMV significantly induced α3 in human foreskin fibroblasts, while MCMV did not induce mouse α3. Infection with guinea pig CMV (GPCMV) in GP fibroblasts also induced α3, and CGs inhibited GPCMV replication. HCMV inhibition with digitoxin reduced α3 expression. The concentration-dependent inhibition of HCMV with digitoxin analogs also correlated with α3 expression. Intriguingly, α3 was localized to the nucleus, and changes in its expression during infection and digitoxin treatment were mostly limited to the nucleus. At 4 h post-infection, α3 colocalized with immediate early 1 (IE1) and the promyelocytic leukemia protein (PML). An interaction of α3-PML-IE1 at 24 h post-infection was disrupted by digitoxin. The mRNA levels of IE1, major immediate early promoter (MIEP)-derived IE, and antiviral cytokines were reduced in infected digitoxin-treated cells. Summarized, these findings suggest a new role for α3 in the anti-HCMV activities of CGs via nuclear antiviral signaling pathways. Full article

The transfer time of Atlantic salmon smolts from freshwater to seawater remains a challenge in aquaculture, with the “smolt window” referring to the optimal timeframe for seawater readiness. Our study monitored Atlantic salmon osmoregulatory adaptations during smoltification under continuous light (LL) and winter signal regime (6 weeks LD 12:12) followed by 6 or 8 weeks of constant light. Fish were subsequently reared in seawater for 8 weeks and subjected to a stress event of cyclic hypoxia at the conclusion of the trial. Significant differences in growth trajectories were observed between the LL and LD groups, with fish receiving the winter signal showing compensatory growth after seawater transfer. Gill Na⁺/K⁺-ATPase (NKA) activity, plasma ions, glucose, and cortisol levels confirmed the importance of the winter signal for seawater adaptation. Molecular markers, including nka isoforms, Na⁺-K⁺-2Cl⁻ cotransporter (nkcc), cystic fibrosis transmembrane conductance regulator (cftr), and Na⁺/HCO₃⁻ cotransporter (nbc), showed distinct temporal expression patterns, particularly in gills and midgut. Notably, the LD group with delayed seawater transfer exhibited enhanced growth and improved hypo-osmoregulatory capacity. These findings underscore the advantages of a winter signal in smoltification and suggest that delaying seawater transfer for up to 8 weeks could be beneficial. Full article

Sertraline, a selective serotonin reuptake inhibitor (SSRI), is commonly used to treat various psychiatric disorders such as depression and anxiety due to its ability to increase serotonin availability in the brain. Recent findings suggest that sertraline may also influence the expression of genes related to synaptic plasticity and neuronal signaling pathways. Alternative splicing, a process that allows a single gene to produce multiple protein isoforms, plays a crucial role in the regulation of neuronal functions and plasticity. Dysregulation of alternative splicing events has been linked to various neurodevelopmental and neurodegenerative diseases. This study aims to explore the effects of sertraline on alternative splicing events, including exon inclusion, exon exclusion, and mutually exclusive splicing events, in genes associated with neuronal function in Drosophila melanogaster and to use this model to investigate the molecular impacts of SSRIs on gene regulation in the nervous system. RNA sequencing (RNA-seq) was performed on central nervous system samples from Drosophila melanogaster adults exposed to sertraline for 24 h when they were third instar larvae. Alternative splicing events were analyzed to identify changes in exon inclusion and exclusion, as well as intron retention. Sertraline treatment significantly altered alternative splicing patterns in key genes related to neuronal stability and function. Specifically, sertraline promoted the inclusion of long Ank2 isoforms, suggesting enhanced axonal stability, and favored long ATPalpha isoforms, which support Na⁺/K⁺ ATPase activity essential for ionic balance and neuronal excitability. Intron retention in the yuri gene suggests that cytoskeletal reorganization could impact neuronal morphology. Additionally, splicing alterations in sxc and Atg18a indicate a potential influence of sertraline on epigenetic regulation and autophagy processes, fundamental aspects for neuronal plasticity and cellular homeostasis. These findings suggest that sertraline influences alternative splicing in the central nervous system of Drosophila melanogaster, potentially contributing to its therapeutic effects by modulating neuronal stability and adaptability. Full article

Na⁺,K⁺-ATPase is the active ion transport system that maintains the electrochemical gradients for Na⁺ and K⁺ across the plasma membrane of most animal cells. Na⁺,K⁺-ATPase is constituted by the association of two major subunits, a catalytic α and a glycosylated β subunit, both of which exist as different isoforms (in mammals known as α1, α2, α3, α4, β1, β2 and β3). Na⁺,K⁺-ATPase α and β isoforms assemble in different combinations to produce various isozymes with tissue specific expression and distinct biochemical properties. Na⁺,K⁺-ATPase α4β1 is only found in male germ cells of the testis and is mainly expressed in the sperm flagellum, where it plays a critical role in sperm motility and male fertility. Here, we report the molecular structure of Na⁺,K⁺-ATPase α4β1 at 2.37 Å resolution in the ouabain-bound state and in the presence of beryllium fluoride. Overall, Na⁺,K⁺-ATPase α4 structure exhibits the basic major domains of a P-Type ATPase, resembling Na⁺,K⁺-ATPase α1, but has differences specific to its distinct sequence. Dissimilarities include the site where the inhibitor ouabain binds. Molecular simulations indicate that glycosphingolipids can bind to a putative glycosphingolipid binding site, which could potentially modulate Na⁺,K⁺-ATPase α4 activity. This is the first experimental evidence for the structure of Na⁺,K⁺-ATPase α4β1. These data provide a template that will aid in better understanding the function Na⁺,K⁺-ATPase α4β1 and will be important for the design and development of compounds that can modulate Na⁺,K⁺-ATPase α4 activity for the purpose of improving male fertility or to achieve male contraception. Full article

Na⁺-K⁺ ATPase is an integral component of cardiac sarcolemma and consists of three major subunits, namely the α-subunit with three isoforms (α₁, α₂, and α₃), β-subunit with two isoforms (β₁ and β₂) and γ-subunit (phospholemman). This enzyme has been demonstrated to transport three Na and two K ions to generate a trans-membrane gradient, maintain cation homeostasis in cardiomyocytes and participate in regulating contractile force development. Na⁺-K⁺ ATPase serves as a receptor for both exogenous and endogenous cardiotonic glycosides and steroids, and a signal transducer for modifying myocardial metabolism as well as cellular survival and death. In addition, Na⁺-K⁺ ATPase is regulated by different hormones through the phosphorylation/dephosphorylation of phospholemman, which is tightly bound to this enzyme. The activity of Na⁺-K⁺ ATPase has been reported to be increased, unaltered and depressed in failing hearts depending upon the type and stage of heart failure as well as the association/disassociation of phospholemman and binding with endogenous cardiotonic steroids, namely endogenous ouabain and marinobufagenin. Increased Na⁺-K⁺ ATPase activity in association with a depressed level of intracellular Na⁺ in failing hearts is considered to decrease intracellular Ca²⁺ and serve as an adaptive mechanism for maintaining cardiac function. The slight to moderate depression of Na⁺-K⁺ ATPase by cardiac glycosides in association with an increased level of Na⁺ in cardiomyocytes is known to produce beneficial effects in failing hearts. On the other hand, markedly reduced Na⁺-K⁺ ATPase activity associated with an increased level of intracellular Na⁺ in failing hearts has been demonstrated to result in an intracellular Ca²⁺ overload, the occurrence of cardiac arrhythmias and depression in cardiac function during the development of heart failure. Furthermore, the status of Na⁺-K⁺ ATPase activity in heart failure is determined by changes in isoform subunits of the enzyme, the development of oxidative stress, intracellular Ca²⁺-overload, protease activation, the activity of inflammatory cytokines and sarcolemmal lipid composition. Evidence has been presented to show that marked alterations in myocardial cations cannot be explained exclusively on the basis of sarcolemma alterations, as other Ca²⁺ channels, cation transporters and exchangers may be involved in this event. A marked reduction in Na⁺-K⁺ ATPase activity due to a shift in its isoform subunits in association with intracellular Ca²⁺-overload, cardiac energy depletion, increased membrane permeability, Ca²⁺-handling abnormalities and damage to myocardial ultrastructure appear to be involved in the progression of heart failure. Full article

Pulsed field ablation (PFA) is a promising new treatment for atrial fibrillation (AF), in which pulmonary vein isolation is achieved by irreversible electroporation. Electroporation causes ATP to leak through the permeabilized membrane. ATP is required both for the healing of the cell membrane and for the functioning of ion pumps, such as sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) or Na⁺,K⁺-ATPase (NKA), which play a key role in maintaining continuous contractions of the heart muscle. We investigated the effects of electroporation on the expression of ion pumps and possible correlations with the activation of AMPK, the main energy sensor in cells. H9c2 rat cardiac cells were exposed to either monopolar or bipolar (H-FIRE) pulses. Cells lysed 4 or 24 h after electroporation were used for mRNA and protein expression analyses. Overall, both pulse protocols caused a dose-dependent downregulation of crucial SERCA and NKA isoforms, except for NKAα2 and β3, which were upregulated after 24 h. Monopolar pulses also decreased the phosphorylation of FXYD1, which may cause an inhibition of NKA activity. Both pulse protocols caused an increased AMPK activity, which may decrease both SERCA and NKA activity via calcium/calmodulin-dependent protein kinase. Our results provide important new insights into what happens in surviving cardiomyocytes after they are exposed to PFA. Full article

Bipolar disorder (BD) is a severe and common chronic mental illness characterized by recurrent mood swings between depression and mania. The biological basis of the disease is poorly understood, and its treatment is unsatisfactory. Na⁺, K⁺-ATPase is a major plasma membrane transporter and signal transducer. The catalytic α subunit of this enzyme is the binding site for cardiac steroids. Three α isoforms of the Na⁺, K⁺-ATPase are present in the brain. Previous studies have supported the involvement of the Na⁺, K⁺-ATPase and endogenous cardiac steroids (ECS) in the etiology of BD. Decreased brain ECS has been found to elicit anti-manic and anti-depressive-like behaviors in mice and rats. However, the identity of the specific α isoform involved in these behavioral effects is unknown. Here, we demonstrated that decreasing ECS through intracerebroventricular (i.c.v.) administration of anti-ouabain antibodies (anti-Ou-Ab) decreased the activity of α1^+/− mice in forced swimming tests but did not change the activity in wild type (wt) mice. This treatment also affected exploratory and anxiety behaviors in α1^+/− but not wt mice, as measured in open field tests. The i.c.v. administration of anti-Ou-Ab decreased brain ECS and increased brain Na⁺, K⁺-ATPase activity in wt and α1^+/− mice. The serum ECS was lower in α1^+/− than wt mice. In addition, a study in human participants demonstrated that serum ECS significantly decreased after treatment. These results suggest that the Na⁺, K⁺-ATPase α1 isoform is involved in depressive- and manic-like behaviors and support that the Na⁺, K⁺-ATPase/ECS system participates in the etiology of BD. Full article

The Na,K-ATPase (NKA) pump plays essential roles for optimal function of the heart. NKA activity decreases in necropsy materials from ischemic heart disease, heart failure and in experimental models. Cellular adaptation to hypoxia is regulated by hypoxia-induced transcription factors (HIF); we tested whether HIFs are involved in regulating the expression and intracellular dynamics of the α2-isoform of NKA (α2-NKA). HIF-1α and HIF-2α expression was suppressed in H9c2 cardiomyocytes by adenoviral infection, where cells were kept in 1% O₂ for 24 h. The silencing efficiency of HIFs was tested on the mRNA and protein expression. We measured the mRNA expression of α2-NKA in HIF-silenced and hypoxia-exposed cells. The membrane and intracellular expression of α2-NKA was measured after labelling the cell surface with NHS-SS-biotin, immunoprecipitation and Western blotting. Hypoxia increased the mRNA expression of α2-NKA 5-fold compared to normoxic cells in an HIF-2α-sensitive manner. The plasma membrane expression of α2-NKA increased in hypoxia by 2-fold and was fully prevented by HIF-2α silencing. Intracellular expression of α2-NKA was not affected. These results showed for the first time that in hypoxic cardiomyocytes α2-NKA is transcriptionally and translationally regulated by HIF-2α. The molecular mechanism behind this regulation needs further investigation. Full article

The optimal function of the Na,K-ATPase (NKA) pump is essential for the heart. In ischemic heart disease, NKA activity decreases due to the decreased expression of the pump subunits. Here, we tested whether the hypoxia-inducible transcription factor (HIF-1α), the key signaling molecule regulating the adaptation of cells to hypoxia, is involved in controlling the expression and cellular dynamics of α1- and β1-NKA isoforms and of NKA activity in in-vitro hypoxic H9c2 cardiomyoblasts. HIF-1α was silenced through adenoviral infection, and cells were kept in normoxia (19% O₂) or hypoxia (1% O₂) for 24 h. We investigated the mRNA and protein expression of α1-, β1-NKA using RT-qPCR and Western blot in whole-cell lysates, cell membranes, and cytoplasmic fractions after labeling the cell surface with NHS-SS-biotin and immunoprecipitation. NKA activity and intracellular ATP levels were also measured. We found that in hypoxia, silencing HIF-1α prevented the decreased mRNA expression of α1-NKA but not of β1-NKA. Hypoxia decreased the plasma membrane expression of α1-NKA and β1- NKA compared to normoxic cells. In hypoxic cells, HIF-1α silencing prevented this effect by inhibiting the internalization of α1-NKA. Total protein expression was not affected. The decreased activity of NKA in hypoxic cells was fully prevented by silencing HIF-1α independent of cellular ATP levels. This study is the first to show that in hypoxic H9c2 cardiomyoblasts, HIF-1α controls the internalization and membrane insertion of α1-NKA subunit and of NKA activity. The mechanism behind this regulation needs further investigation. Full article

The Na⁺-activated Na⁺ channel (Nax) and salt-inducible kinase (SIK) are stimulated by increases in local Na⁺ concentration, affecting (Na⁺ + K⁺)-ATPase activity. To test the hypothesis that the triad Nax/SIK/(Na⁺ + K⁺)-ATPase contributes to kidney injury and salt-sensitive hypertension (HTN), uninephrectomized male Wistar rats (200 g; n = 20) were randomly divided into 4 groups based on a salt diet (normal salt diet; NSD—0.5% NaCl—or high-salt diet; HSD—4% NaCl) and subcutaneous administration of saline (0.9% NaCl) or deoxycorticosterone acetate (DOCA, 8 mg/kg), as follows: Control (CTRL), CTRL-Salt, DOCA, and DOCA-Salt, respectively. After 28 days, the following were measured: kidney function, blood pressure, (Na⁺ + K⁺)-ATPase and SIK1 kidney activities, and Nax and SIK1 renal expression levels. SIK isoforms in kidneys of CTRL rats were present in the glomerulus and tubular epithelia; they were not altered by HSD and/or HTN. CTRL-Salt rats remained normotensive but presented slight kidney function decay. HSD rats displayed augmentation of the Nax/SIK/(Na⁺ + K⁺)-ATPase pathway. HTN, kidney injury, and kidney function decay were present in all DOCA rats; these were aggravated by HSD. DOCA rats presented unaltered (Na⁺ + K⁺)-ATPase activity, diminished total SIK activity, and augmented SIK1 and Nax content in the kidney cortex. DOCA-Salt rats expressed SIK1 activity and downregulation in (Na⁺ + K⁺)-ATPase activity in the kidney cortex despite augmented Nax content. The data of this study indicate that the (Na⁺ + K⁺)-ATPase activity response to SIK is attenuated in rats under HSD, independent of HTN, as a mechanism contributing to kidney injury and salt-sensitive HTN. Full article

Two α-isoforms of the Na⁺,K⁺-ATPase (α₁ and α₂) are expressed in the cardiovascular system, and it is unclear which isoform is the preferential regulator of contractility. Mice heterozygous for the familial hemiplegic migraine type 2 (FHM2) associated mutation in the α₂-isoform (G301R; α₂^+/G301R mice) have decreased expression of cardiac α₂-isoform but elevated expression of the α₁-isoform. We aimed to investigate the contribution of the α₂-isoform function to the cardiac phenotype of α₂^+/G301R hearts. We hypothesized that α₂^+/G301R hearts exhibit greater contractility due to reduced expression of cardiac α₂-isoform. Variables for contractility and relaxation of isolated hearts were assessed in the Langendorff system without and in the presence of ouabain (1 µM). Atrial pacing was performed to investigate rate-dependent changes. The α₂^+/G301R hearts displayed greater contractility than WT hearts during sinus rhythm, which was rate-dependent. The inotropic effect of ouabain was more augmented in α₂^+/G301R hearts than in WT hearts during sinus rhythm and atrial pacing. In conclusion, cardiac contractility was greater in α₂^+/G301R hearts than in WT hearts under resting conditions. The inotropic effect of ouabain was rate-independent and enhanced in α₂^+/G301R hearts, which was associated with increased systolic work. Full article

A lipopolysaccharide (LPS)-induced neuroinflammation rat model was used to study the effects of ouabain (OUA) at low concentrations, which can interact with the Na,K-ATPase, causing the modulation of intracellular signalling pathways in the Central Nervous System. Our study aimed to analyse the effects of OUA on glutamate transport in the hippocampus of rats with LPS-induced neuroinflammation. Adult male Wistar rats were divided into four groups: OUA (1.8 µg/kg), saline (CTR), LPS (200 µg/kg), and OUA + LPS (OUA 20 min before LPS). The animals were sacrificed after 2 h, and the hippocampus was collected for analysis. After treatment, we determined the activities of Na,K-ATPase and glutamine synthetase (GS). In addition, expression of the α1, α2, and α3 isoforms of Na,K-ATPase and the glutamate transporters, EAAT1 and EAAT2, were also analysed. Treatment with OUA caused a specific increase in the α2 isoform expression (~20%), whereas LPS decreased its expression (~22%), and treatment with OUA before LPS prevented the effects of LPS. Moreover, LPS caused a decrease of approximately 50% in GS activity compared with that in the CTR group; however, OUA pre-treatment attenuated this effect of LPS. Notably, it was found that treatment with OUA caused an increase in the expression of EAAT1 (~30%) and EAAT2 (~25%), whereas LPS caused a decrease in the expression of EAAT1 (~23%) and EAAT2 (~25%) compared with that in the CTR group. When treated with OUA, the effects of LPS were abrogated. In conclusion, the OUA pre-treatment abolished the effect caused by LPS, suggesting that this finding may be related to the restoration of the interaction between FXYD2 and the studied membrane proteins. Full article

Na⁺/K⁺ ATPase (NKA) comprises several subunits to provide isozyme heterogeneity in a tissue-specific manner. An abundance of NKA α, β, and FXYD1 subunits is well-described in human skeletal muscle, but not much is known about FXYD5 (dysadherin), a regulator of NKA and β1 subunit glycosylation, especially with regard to fibre-type specificity and influence of sex and exercise training. Here, we investigated muscle fibre-type specific adaptations in FXYD5 and glycosylated NKAβ1 to high-intensity interval training (HIIT), as well as sex differences in FXYD5 abundance. In nine young males (23.8 ± 2.5 years of age) (mean ± SD), 3 weekly sessions of HIIT for 6 weeks enhanced muscle endurance (220 ± 102 vs. 119 ± 99 s, p < 0.01) and lowered leg K⁺ release during intense knee-extensor exercise (0.5 ± 0.8 vs. 1.0 ± 0.8 mmol·min^–1, p < 0.01) while also increasing cumulated leg K⁺ reuptake 0–3 min into recovery (2.1 ± 1.5 vs. 0.3 ± 0.9 mmol, p < 0.01). In type IIa muscle fibres, HIIT lowered FXYD5 abundance (p < 0.01) and increased the relative distribution of glycosylated NKAβ1 (p < 0.05). FXYD5 abundance in type IIa muscle fibres correlated inversely with the maximal oxygen consumption (r = –0.53, p < 0.05). NKAα2 and β1 subunit abundances did not change with HIIT. In muscle fibres from 30 trained males and females, we observed no sex (p = 0.87) or fibre type differences (p = 0.44) in FXYD5 abundance. Thus, HIIT downregulates FXYD5 and increases the distribution of glycosylated NKAβ1 in type IIa muscle fibres, which is likely independent of a change in the number of NKA complexes. These adaptations may contribute to counter exercise-related K⁺ shifts and enhance muscle performance during intense exercise. Full article