Structure and Mechanism of P-Type ATPase Ion Pumps.

P-type ATPases are found in all kingdoms of life and constitute a wide range of cation transporters, primarily for H+, Na+, K+, Ca2+, and transition metal ions such as Cu(I), Zn(II), and Cd(II). They have been studied through a wide range of techniques, and research has gained very significant insight on their transport mechanism and regulation. Here, we review the structure, function, and dynamics of P2-ATPases including Ca2+-ATPases and Na,K-ATPase. We highlight mechanisms of functional transitions that are associated with ion exchange on either side of the membrane and how the functional cycle is regulated by interaction partners, autoregulatory domains, and off-cycle states. Finally, we discuss future perspectives based on emerging techniques and insights.

Molecular determinants for cold adaptation in an Antarctic Na+/K+-ATPase.

Enzymes from ectotherms living in chronically cold environments have evolved structural innovations to overcome the effects of temperature on catalysis. Cold adaptation of soluble enzymes is driven by changes within their primary structure or the aqueous milieu. For membrane-embedded enzymes, like the Na+/K+-ATPase, the situation is different because changes to the lipid bilayer in which they operate may also be relevant. Although much attention has been focused on thermal adaptation within lipid bilayers, relatively little is known about the contribution of structural changes within membrane-bound enzymes themselves. The identification of specific mutations that confer temperature compensation is complicated by the presence of neutral mutations, which can be more numerous. In the present study, we identified specific amino acids in a Na+/K+-ATPase from an Antarctic octopus that underlie cold resistance. Our approach was to generate chimeras between an Antarctic clone and a temperate ortholog and then study their temperature sensitivities in Xenopus oocytes using an electrophysiological approach. We identified 12 positions in the Antarctic Na+/K+-ATPase that, when transferred to the temperate ortholog, were sufficient to confer cold tolerance. Furthermore, although all 12 Antarctic mutations were required for the full phenotype, a single leucine in the third transmembrane segment (M3) imparted most of it. Mutations that confer cold resistance are mostly in transmembrane segments, at positions that face the lipid bilayer. We propose that the interface between a transmembrane enzyme and the lipid bilayer is a critical determinant of temperature sensitivity and, accordingly, has been a prime evolutionary target for thermal adaptation.

A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

Prostaglandin E2 activates melanin-concentrating hormone neurons to drive diet-induced obesity.

Hypothalamic inflammation reduces appetite and body weight during inflammatory diseases, while promoting weight gain when induced by high-fat diet (HFD). How hypothalamic inflammation can induce opposite energy balance outcomes remains unclear. We found that prostaglandin E2 (PGE2), a key hypothalamic inflammatory mediator of sickness, also mediates diet-induced obesity (DIO) by activating appetite-promoting melanin-concentrating hormone (MCH) neurons in the hypothalamus in rats and mice. The effect of PGE2 on MCH neurons is excitatory at low concentrations while inhibitory at high concentrations, indicating that these neurons can bidirectionally respond to varying levels of inflammation. During prolonged HFD, endogenous PGE2 depolarizes MCH neurons through an EP2 receptor-mediated inhibition of the electrogenic Na+/K+-ATPase. Disrupting this mechanism by genetic deletion of EP2 receptors on MCH neurons is protective against DIO and liver steatosis in male and female mice. Thus, an inflammatory mediator can directly stimulate appetite-promoting neurons to exacerbate DIO and fatty liver.

Vascular smooth muscle BK channels limit ouabain-induced vasocontraction: Dual role of the Na/K-ATPase as a hub for Src-kinase and the Na/Ca-exchanger.

Large-conductance, calcium-activated potassium channels (BK channels) and the Na/K-ATPase are expressed universally in vascular smooth muscle. The Na/K-ATPase may act via changes in the intracellular Ca2+ concentration mediated by the Na/Ca exchanger (NCX) and via Src kinase. Both pathways are known to regulate BK channels. Whether BK channels functionally interact in vascular smooth muscle cells with the Na/K-ATPase remains to be elucidated. Thus, this study addressed the hypothesis that BK channels limit ouabain-induced vasocontraction. Rat mesenteric arteries were studied using isometric myography, FURA-2 fluorimetry and proximity ligation assay. The BK channel blocker iberiotoxin potentiated methoxamine-induced contractions. The cardiotonic steroid, ouabain (10-5 M), induced a contractile effect of IBTX at basal tension prior to methoxamine administration and enhanced the pro-contractile effect of IBTX on methoxamine-induced contractions. These facilitating effects of ouabain were prevented by the inhibition of either NCX or Src kinase. Furthermore, inhibition of NCX or Src kinase reduced the BK channel-mediated negative feedback regulation of arterial contraction. The effects of NCX and Src kinase inhibition were independent of each other. Co-localization of the Na/K-ATPase and the BK channel was evident. Our data suggest that BK channels limit ouabain-induced vasocontraction by a dual mechanism involving the NCX and Src kinase signaling. The data propose that the NCX and the Src kinase pathways, mediating the ouabain-induced activation of the BK channel, act in an independent manner.

Cryoelectron microscopy of Na+,K+-ATPase in the two E2P states with and without cardiotonic steroids.

Cryoelectron microscopy (cryo-EM) was applied to Na+,K+-ATPase (NKA) to determine the structures of two E2P states, one (E2PATP) formed by ATP and Mg2+ in the forward reaction, and the other (E2PPi) formed by inorganic phosphate (Pi) and Mg2+ in the backward reaction, with and without ouabain or istaroxime, representatives of classical and new-generation cardiotonic steroids (CTSs). These two E2P states exhibit different biochemical properties. In particular, K+-sensitive acceleration of the dephosphorylation reaction is not observed with E2PPi, attributed to the presence of a Mg2+ ion in the transmembrane cation binding sites. The cryo-EM structures of NKA demonstrate that the two E2P structures are nearly identical but Mg2+ in the transmembrane binding cavity is identified only in E2PPi, corroborating the idea that it should be denoted as E2PPi·Mg2+. We can now explain why the absence of transmembrane Mg2+ in E2PATP confers the K+ sensitivity in dephosphorylation. In addition, we show that ATP bridges the actuator (A) and nucleotide binding (N) domains, stabilizing the E2PATP state; CTS binding causes hardly any changes in the structure of NKA, both in E2PATP and E2PPi·Mg2+, indicating that the binding mechanism is conformational selection; and istaroxime binds to NKA, extending its aminoalkyloxime group deep into the cation binding site. This orientation is upside down compared to that of classical CTSs with respect to the steroid ring. Notably, mobile parts of NKA are resolved substantially better in the electron microscopy (EM) maps than in previous X-ray structures, including sugars sticking out from the ß-subunit and many phospholipid molecules.

Role of Na/K-ATPase α1 caveolin-binding motif in adipogenesis.

Deficiencies in mice and in humans have brought to the fore the importance of the caveolar network in key aspects of adipocyte biology. The conserved N-terminal caveolin-binding motif (CBM) of the ubiquitous Na/K-ATPase (NKA) α1 isoform, which allows NKA/caveolin-1 (Cav1) interaction, influences NKA signaling and caveolar distribution. It has been shown to be critical for animal development and ontogenesis, as well as lineage-specific differentiation of human induced pluripotent stem cells (hiPSCs). However, its role in postnatal adipogenesis has not been fully examined. Using a genetic approach to alter CBM in hiPSC-derived adipocytes (iAdi-mCBM) and in mice (mCBM), we investigated the regulatory function of NKA CBM signaling in adipogenesis. Seahorse XF cell metabolism analyses revealed impaired glycolysis and decreased ATP synthesis-coupled respiration in iAdi-mCBM. These metabolic dysfunctions were accompanied by evidence of extensive remodeling of the extracellular matrix (ECM), including increased collagen staining, overexpression of ECM marker genes, and heightened TGF-ß signaling uncovered by RNAseq analysis. Rescue of mCBM by lentiviral delivery of WT NKA α1 or treatment of mCBM hiPSCs with the TGF-ß inhibitor SB431542 normalized ECM, suggesting that NKA CBM signaling integrity is required for adequate control of TGF-ß signaling and ECM stiffness during adipogenesis. The physiological impact was revealed in mCBM male mice with reduced fat mass accompanied by histological and transcriptional evidence of elevated adipose fibrosis and decreased adipocyte size. Based on these findings, we propose that the genetic alteration of the NKA/Cav1 regulatory path uncovered in human iAdi leads to lipodystrophy in mice.NEW & NOTEWORTHY A Na/K-ATPase α1 caveolin-binding motif regulates adipogenesis. Mutation of this binding motif in the mouse leads to reduced fat with increased extracellular matrix production and inflammation. RNA-seq analysis and pharmacological interventions in human iPSC-derived adipocytes revealed that TGF-ß signal, rather than Na/K-ATPase-mediated ion transport, is a key mediator of NKA regulation of adipogenesis.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.

Resting membrane potential and intracellular [Na+] at rest, during fatigue and during recovery in rat soleus muscle fibres in situ.

Large trans-sarcolemmal ionic shifts occur with fatiguing exercise or stimulation of isolated muscles. However, it is unknown how resting membrane potential (EM) and intracellular sodium concentration ([Na+]i) change with repeated contractions in living mammals. We investigated (i) whether [Na+]i (peak, kinetics) can reveal changes of Na+-K+ pump activity during brief or fatiguing stimulation and (ii) how resting EM and [Na+]i change during fatigue and recovery of rat soleus muscle in situ. Muscles of anaesthetised rats were stimulated with brief (10 s) or repeated tetani (60 Hz for 200 ms, every 2 s, for 30 s or 300 s) with isometric force measured. Double-barrelled ion-sensitive microelectrodes were used to quantify resting EM and [Na+]i. Post-stimulation data were fitted using polynomials and back-extrapolated to time zero recovery. Mean pre-stimulation resting EM (layer 2-7 fibres) was -71 mV (surface fibres were more depolarised), and [Na+]i was 14 mM. With deeper fibres, 10 s stimulation (2-150 Hz) increased [Na+]i to 38-46 mM whilst simultaneously causing hyperpolarisations (7.3 mV for 2-90 Hz). Fatiguing stimulation for 30 s or 300 s led to end-stimulation resting EM of -61 to -53 mV, which recovered rapidly (T1/2, 8-22 s). Mean end-stimulation [Na+]i increased to 86-101 mM with both fatigue protocols and the [Na+]i recovery time-course (T1/2, 21-35 s) showed no difference between protocols. These combined findings suggest that brief stimulation hyperpolarises the resting EM, likely via maximum Na+-induced stimulation of the Na+-K+ pump. Repeated tetani caused massive depolarisation and elevations of [Na+]i that together lower force, although they likely interact with other factors to cause fatigue. [Na+]i recovery kinetics provided no evidence of impaired Na+-K+ pump activity with fatigue. KEY POINTS: It is uncertain how resting membrane potential, intracellular sodium concentration ([Na+]i), and sodium-potassium (Na+-K+) pump activity change during repeated muscle contractions in living mammals. For rat soleus muscle fibres in situ, brief tetanic stimulation for 10 s led to raised [Na+]i, anticipated to evoke maximal Na+-induced stimulation of the Na+-K+ pump causing an immediate hyperpolarisation of the sarcolemma. More prolonged stimulation with repeated tetanic contractions causes massive elevations of [Na+]i, which together with large depolarisations (via K+ disturbances) likely reduce force production. These effects occurred without impairment of Na+-K+ pump function. Together these findings suggest that rapid activation of the Na+-K+ pump occurs with brief stimulation to maintain excitability, whereas more prolonged stimulation causes rundown of the trans-sarcolemmal K+ gradient (hence depolarisation) and Na+ gradient, which in combination can impair contraction to contribute to fatigue in living mammals.

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. For arginine 756, at the junction of membrane and cytoplasmic domains, mutations produce encephalopathy during febrile infections. Here we tested the pathogenicity of p.Arg756His (R756H) in isogenic mammalian cells. R756H protein had sufficient transport activity to support cells when endogenous ATP1A1 was inhibited. It had half the turnover rate of wildtype, reduced affinity for Na+, and increased affinity for K+. There was modest endoplasmic reticulum retention during biosynthesis at 37 °C but little benefit from the folding drug phenylbutyrate (4-PBA), suggesting a tolerated level of misfolding. When cells were incubated at just 39 °C, however, α3 protein level dropped without loss of ß subunit, paralleled by an increase of endogenous α1. Elevated temperature resulted in internalization of α3 from the surface along with some ß subunit, accompanied by cytoplasmic redistribution of a marker of lysosomes and endosomes, lysosomal-associated membrane protein 1. After return to 37 °C, α3 protein levels recovered with cycloheximide-sensitive new protein synthesis. Heating in vitro showed activity loss at a rate 20- to 30-fold faster than wildtype, indicating a temperature-dependent destabilization of protein structure. Arg756 appears to confer thermal resistance as an anchor, forming hydrogen bonds among four linearly distant parts of the Na,K-ATPase structure. Taken together, our observations are consistent with fever-induced symptoms in patients.

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport.

The Na+/K+-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na+ for extracellular K+ to the hydrolysis of ATP. The asymmetric distribution of Na+ and K+ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers-Post model. It involves the presence of gates alternatively exposing Na+/K+-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K+, information is lacking about Na+-occluded intermediates, as occluded Na+ was only detected in states incapable of performing a catalytic cycle, including two Na+-containing crystallographic structures. The current knowledge is that intracellular Na+ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na+-occluded intermediates, we isolated species with tightly bound Na+ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na+ becomes spontaneously occluded in the E1 dephosphorylated form of the Na+/K+-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na+ occlusion as it would have been expected; on the contrary, occluded Na+ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers-Post model for explaining the Na+ transport pathway.

The lethal homozygous variant in the ATP1A2 gene is associated with FARIMPD syndrome phenotypes in newborns.

FARIMPD (Fetal akinesia, respiratory insufficiency, microcephaly, polymicrogyria, and dysmorphic facies) syndrome is a severe condition caused by ATP1A2 gene variants. The syndrome's novelty and rarity have limited its clinical and molecular knowledge. This research tries to provide new insight by investigating the cause of the early deaths due to FARIMPD syndrome in a particular family and reviewing previous studies. DNA and RNA were extracted from the blood samples of newborns and their parents, followed by whole exome sequencing and segregation analysis. A pathogenic homozygous nonsense variant (c.1234C > T: p.Arg412*) in the ATP1A2 gene was found in newborns. This variant is reported as homozygous for the first time. The migraine symptoms were the result of the heterozygous state of this particular variant, which supported the dominant inheritance pattern of this disease. Real-time PCR was used to analyze ATP1A2 gene expression in the newborns compared to parents and control subjects. The expression analysis also showed significant mRNA degradation in the newborns compared to heterozygous and healthy individuals, due to Nonsense-mediated mRNA Decay phenomena. Our study describes an ATP1A2 nonsense variant (c.1234C > T) that appears compatible with infant survival in the heterozygous and compound heterozygous states but is lethal in the homozygous state.

Spontaneous spreading depolarizations originate subcortically in a novel mouse model of familial hemiplegic migraine type 2.

The mechanisms of initiation of spreading depolarization (SD) are understudied due to a paucity of disease models with spontaneously occurring events. We here present a novel mouse model of familial hemiplegic migraine type 2 (FHM2), expressing the missense T345A-mutated α2 subunit of the Na+/K+ adenosine triphosphatase pump (Atp1a2T345A). Homozygous Atp1a2T345A mice showed regular spontaneous SDs that exhibit a diurnal rhythm and typically originate from the hippocampus. Heterozygous Atp1a2T345A mice rarely exhibited spontaneous SDs and, for electrically induced SDs, only showed an increased propagation speed, whereas homozygotes showed both increased propagation and decreased threshold. Remarkably, despite hippocampal hyperexcitability, spontaneous SDs in Atp1a2T345A mice were only rarely associated with epileptic behavior, and seizure expression during kindling was decreased. Spontaneous SDs could be prevented by modulation of persistent sodium currents. Hippocampal SDs occurred in the presence of an NMDA-receptor antagonist, but these events did not reach the cortex, suggesting that initiation and propagation of SD depend on different mechanisms in this model.

The effect of cytochrome c on Na,K-ATPase.

Na,K-ATPase is a crucial enzyme responsible for maintaining Na+, K+-gradients across the cell membrane, which is essential for numerous physiological processes within various organs and tissues. Due to its significance in cellular physiology, inhibiting Na,K-ATPase can have profound physiological consequences. This characteristic makes it a target for various pharmacological applications, and drugs that modulate the pump's activity are thus used in the treatment of several medical conditions. Cytochrome c (Cytc) is a protein with dual functions in the cell. In the mitochondria, it is essential for ATP synthesis and energy production. However, in response to apoptotic stimuli, it is released into the cytosol, where it triggers programmed cell death through the intrinsic apoptosis pathway. Aside from its role in canonical intrinsic apoptosis, Cytc also plays additional roles. For instance, Cytc participates in certain non-apoptotic functions -those which are less well-understood in comparison to its role in apoptosis. Within this in vitro study, we have shown the impact of Cytc on Na,K-ATPase for the first time. Cytc has a biphasic action on Na,K-ATPase, with activation at low concentrations (0.06 ng/ml; 6 ng/ml) and inhibition at high concentration (120 ng/ml). Cytc moreover displays isoform/subunit specificity and regulates the Na+ form of the enzyme, while having no effect on the activity or kinetic parameters of the K+-dependent form of the enzyme. Changing the affinity of p-chloromercuribenzoic acid (PCMB) by Cytc is therefore both a required and sufficient condition for confirming that PCMB and Cytc share the same target, namely the thiol groups of cysteine in Na,K-ATPase.

Integrated organismal responses induced by projected levels of CO2 and temperature exposures in the early life stages of lake sturgeon.

Atmospheric CO2 and temperature are rising concurrently, and may have profound impacts on the transcriptional, physiological and behavioural responses of aquatic organisms. Further, spring snowmelt may cause transient increases of pCO2 in many freshwater systems. We examined the behavioural, physiological and transcriptomic responses of an ancient fish, the lake sturgeon (Acipenser fulvescens) to projected levels of warming and pCO2 during its most vulnerable period of life, the first year. Specifically, larval fish were raised in either low (16°C) or high (22°C) temperature, and/or low (1000 µatm) or high (2500 µatm) pCO2 in a crossed experimental design over approximately 8 months. Following overwintering, lake sturgeon were exposed to a transient increase in pCO2 of 10,000 µatm, simulating a spring melt based on data in freshwater systems. Transcriptional analyses revealed potential connections to otolith formation and reduced growth in fish exposed to high pCO2 and temperature in combination. Network analyses of differential gene expression revealed different biological processes among the different treatments on the edges of transcriptional networks. Na+/K+-ATPase activity increased in fish not exposed to elevated pCO2 during development, and mRNA abundance of the ß subunit was most strongly predictive of enzyme activity. Behavioural assays revealed a decrease in total activity following an acute CO2 exposure. These results demonstrate compensatory and compounding mechanisms of pCO2 and warming dependent on developmental conditions in lake sturgeon. Conserved elements of the cellular stress response across all organisms provide key information for how other freshwater organisms may respond to future climate change.

Regulation of Na+-K+-ATPase leads to disturbances of isoproterenol-induced cardiac dysfunction via interference of Ca2+-dependent cardiac metabolism.

Reductions in Na+-K+-ATPase (NKA) activity and expression are often observed in the progress of various reason-induced heart failure (HF). However, NKA α1 mutation or knockdown cannot cause spontaneous heart disease. Whether the abnormal NKA α1 directly contributes to HF pathogenesis remains unknown. Here, we challenge NKA α1+/- mice with isoproterenol to evaluate the role of NKA α1 haploinsufficiency in isoproterenol (ISO)-induced cardiac dysfunction. Genetic knockdown of NKA α1 accelerated ISO-induced cardiac cell hypertrophy, heart fibrosis, and dysfunction. Further studies revealed decreased Krebs cycle, fatty acid oxidation, and mitochondrial OXPHOS in the hearts of NKA α1+/- mice challenged with ISO. In ISO-treated conditions, inhibition of NKA elevated cytosolic Na+, further reduced mitochondrial Ca2+ via mNCE, and then finally down-regulated cardiac cell energy metabolism. In addition, a supplement of DRm217 alleviated ISO-induced heart dysfunction, mitigated cardiac remodeling, and improved cytosolic Na+ and Ca2+ elevation and mitochondrial Ca2+ depression in the NKA α1+/- mouse model. The findings suggest that targeting NKA and mitochondria Ca2+ could be a promising strategy in the treatment of heart disease.

Na/K-ATPase signaling tonically inhibits sodium reabsorption in the renal proximal tubule.

Through its classic ATP-dependent ion-pumping function, basolateral Na/K-ATPase (NKA) generates the Na+ gradient that drives apical Na+ reabsorption in the renal proximal tubule (RPT), primarily through the Na+ /H+ exchanger (NHE3). Accordingly, activation of NKA-mediated ion transport decreases natriuresis through activation of basolateral (NKA) and apical (NHE3) Na+ reabsorption. In contrast, activation of the more recently discovered NKA signaling function triggers cellular redistribution of RPT NKA and NHE3 and decreases Na+ reabsorption. We used gene targeting to test the respective contributions of NKA signaling and ion pumping to the overall regulation of RPT Na+ reabsorption. Knockdown of RPT NKA in cells and mice increased membrane NHE3 and Na+ /HCO3 - cotransporter (NBCe1A). Urine output and absolute Na+ excretion decreased by 65%, driven by increased RPT Na+ reabsorption (as indicated by decreased lithium clearance and unchanged glomerular filtration rate), and accompanied by elevated blood pressure. This hyper reabsorptive phenotype was rescued upon crossing with RPT NHE3-/- mice, confirming the importance of NKA/NHE3 coupling. Hence, NKA signaling exerts a tonic inhibition on Na+ reabsorption by regulating key apical and basolateral Na+ transporters. This action, lifted upon NKA genetic suppression, tonically counteracts NKA's ATP-driven function of basolateral Na+ reabsorption. Strikingly, NKA signaling is not only physiologically relevant but it also appears to be functionally dominant over NKA ion pumping in the control of RPT reabsorption.

AMPK and glucose deprivation exert an isoform-specific effect on the expression of Na+,K+-ATPase subunits in cultured myotubes.

In skeletal muscle, Na+,K+-ATPase (NKA), a heterodimeric (α/ß) P-type ATPase, has an essential role in maintenance of Na+ and K+ homeostasis, excitability, and contractility. AMP-activated protein kinase (AMPK), an energy sensor, increases the membrane abundance and activity of NKA in L6 myotubes, but its potential role in regulation of NKA content in skeletal muscle, which determines maximum capacity for Na+ and K+ transport, has not been clearly delineated. We examined whether energy stress and/or AMPK affect expression of NKA subunits in rat L6 and primary human myotubes. Energy stress, induced by glucose deprivation, increased protein content of NKAα1 and NKAα2 in L6 myotubes, while decreasing the content of NKAα1 in human myotubes. Pharmacological AMPK activators (AICAR, A-769662, and diflunisal) modulated expression of NKA subunits, but their effects only partially mimicked those that occurred in response to glucose deprivation, indicating that AMPK does not mediate all effects of energy stress on NKA expression. Gene silencing of AMPKα1/α2 increased protein levels of NKAα1 in L6 myotubes and NKAα1 mRNA levels in human myotubes, while decreasing NKAα2 protein levels in L6 myotubes. Collectively, our results suggest a role for energy stress and AMPK in modulation of NKA expression in skeletal muscle. However, their modulatory effects were not conserved between L6 myotubes and primary human myotubes, which suggests that coupling between energy stress, AMPK, and regulation of NKA expression in vitro depends on skeletal muscle cell model.

Autosis: a new form of cell death in myocardial ischemia-reperfusion injury.

Cardiomyocytes undergo a variety of cell death events during myocardial ischemia‒reperfusion injury (MIRI). Understanding the causes of cardiomyocyte mortality is critical for the prevention and treatment of MIRI. Among the various types of cell death, autosis is a recently identified type of autophagic cell death with distinct morphological and chemical characteristics. Autosis can be attenuated by autophagy inhibitors but not reversed by apoptosis or necrosis inhibitors. In recent years, it has been shown that during the late phase of reperfusion, autosis is activated, which exacerbates myocardial injury. This article describes the characteristics of autosis, autophagic cell death, and the relationship between autophagic cell death and autosis; reviews the mechanism of autosis in MIRI; and discusses its clinical significance.

Digoxin and its Na+/K+-ATPase-targeted actions on cardiovascular diseases and cancer.

Na+/K+-ATPase (NKA) is a plasma membrane ion-transporting protein involved in the generation and maintenance of Na+ and K+ gradients across the cell membrane, which can produce a driving force for the secondary transport of metabolic substrates. NKA also regulates intracellular calcium that is responsible for modulating numerous cellular processes, while it interacts with many other proteins and functions as a signal transducer, with several signaling pathways being involved. Thus, NKA has become an important target for the treatment of human diseases. Cardiac glycosides are well-known NKA inhibitors, of which (+)-digoxin or digoxin has been long used for the treatment of congestive heart failure. Also, digoxin has exhibited potential antitumor activity, by targeting directly HIF-1α, NKA, and NF-κB. Thus, the function of NKA in human cardiovascular diseases and cancer and the therapeutic effects of digoxin on these diseases are summarized in the present review, with the correlations among digoxin, NKA, cardiovascular diseases, and cancer being discussed. Presented herein are also the antitumor potential of monosaccharide cardiac glycoside analogues of digoxin, including (-)-cryptanoside A, (-)-oleandrin, (-)-ouabain, and (+)-strebloside. It is hoped that this contribution will provide some helpful information for the design and discovery of new cardiac glycoside-type therapeutic agents for the treatment of cardiovascular diseases and cancer.