Reduction in Na+/K+-ATPase levels is associated with obesity, apparently linked to hyperglycemic-hyperinsulinemia, which may repress or inactivate the enzyme. Decrease in cardiac Na+/K+-ATPase activity or protein concentration contributes to the deficiencies in cardiac contractility in animal models and has been documented in patients with heart failure (HF) (Schwinger et al. 2003; Obradovic et al. 2015). The Na+/K+-ATPase is also served as a receptor of digitalis steroids, and after their binding, Na+/K+-ATPase controls myocyte Ca2+ balance and cardiac contractility.
Na+/K+-ATPase belongs to the family of P-type ATPases, also including the closely related H+,K+-ATPase, Ca2+-ATPases, heavy metal-transporting ATPases, and ATPases with lipid substrates and unknown substrates. The common characteristic of these ATPases is the formation of a covalent phosphoenzyme intermediate as a part of the catalytic mechanism. For each ATP hydrolyzed by the Na+/K+-ATPase, three internal Na+ are exchanged for two external K+ in a reaction cycle involving sequential binding and release of Na+ and K+ and transition between two major conformational states, the so-called E1 (Na-selective) and E2 (K-selective) (Therien and Blostein 2000).
The primary effect of the Na+/K+-ATPase is to maintain the low intracellular Na+ and high intracellular K+ concentrations required for a multitude of cellular functions. This occurs in several steps (Therien and Blostein 2000). Following binding of ATP to Na+/K+-ATPase, three Na+ ions from the cytoplasm associate with the molecule. Transfer of a phosphate group (via hydrolysis of ATP) to the Na+/K+-ATPase results in a conformational change that creates an opening at the outside of the cell that allows the three bound Na+ ions to be released. Following cleavage of the phosphate group, two extracellular K+ ions then bind the Na+/K+-ATPase and are released inside the cell (Therien and Blostein 2000; Kaplan 2002). By maintaining the Na+ gradient between intracellular and extracellular compartments, this enzyme has influence on many vital functions, like cell volume, absorption processes in kidney, and excitability in nerve and muscle. Transport of sugars and amino acids in tissues depends on proper activity of Na+/K+-ATPase.
Structure of Na+/K+-ATPase
The Na+/K+-ATPase is a universally expressed transmembrane protein of oligomeric structure, consisting of two main subunits α and β which are present in equal molar ratio (1:1) (Therien and Blostein 2000). In addition, other proteins such as FXYD proteins, also referred as γ subunits, are differently expressed in cell- and tissue-specific manner. The FXYD proteins have a role in stabilizing the Na+/K+-ATPase and regulating its function (Garty and Karlish 2006).
The large α subunit (∼110 kDa) contains 10 transmembrane domains (usually numbered as M1 to M10) and three large intracellular domains. These cytosolic domains catalyze transfer of phosphoryl group. The amino terminal tail and the second cytoplasmic peptide together form actuator domain or A domain required for dephosphorylation of the enzyme and for coupling ATP hydrolysis to ion transport through the membrane (M) domain. The large cytoplasmic loop connecting M4 and M5 forms two domains: the nucleotide-binding domain (N domain) responsible for ATP binding and the phosphorylation domain (P domain) which catalyzes transfer of the c-phosphoryl group to the enzyme (Therien and Blostein 2000; Kaplan 2002).
The smaller β subunit (∼35–55 kDa) is necessary for proper folding and insertion of the newly synthesized protein into the membrane. It is composed from one transmembrane segment, short cytoplasmic tail, and large glycosylated extracellular segment. In addition, β subunit affects the intrinsic ion binding and transport properties of the mature pump. Although β subunit is important for the normal activity of the Na+/K+-ATPase, its role is still not completely understood (Therien and Blostein 2000; Kaplan 2002).
The auxiliary subunit of Na+/K+-ATPase is FXYD family of proteins. Seven different FXYD proteins (FXYD1 to FXYD7) have been identified in mammals. These proteins act as tissue-specific regulatory subunits of the Na+/K+-ATPase kinetic properties. FXYD represent a group of small proteins with a single transmembrane segment containing the invariant FXYD motif (after which they are named) in the extracellular domain, and two conserved glycine and a serine residues. FXYD proteins modify the affinity for Na+, K+, and ATP, influencing kinetics and transport properties of Na+/K+-ATPase. The conserved FXYD motif has an important role in stabilizing interactions between Na+/K+-ATPase and FXYD protein (Garty and Karlish 2006; Geering 2008).
Structurally, α and β isoforms exhibit 85% and 45% identity and display a tissue- and cell-specific distribution and a developmentally regulated pattern of expression. The Na+/K+-ATPase α subunit exists in four isoforms. The α1 isoform is dominant and found in all cells, while α2 and α3 isoforms are present in smaller amounts and are differentially expressed in tissues between species (Geering 2008). In human heart α1, α2, and α3 are expressed together with β1 and very low levels of β2 in a region-specific manner, while in rat heart only α1 and α2 subunits are expressed (Schwinger et al. 2003). The α4 isoform is located in testis and regulates sperm motility (Yan and Shapiro 2016). Three isoforms of β subunits have been identified so far. Similar to the α1 isoform, the β1 isoform is expressed ubiquitously, while β2 is mainly expressed in skeletal muscle and heart, and β3 in testis and central nervous system (Suhail 2010).
The concentration of Na+/K+-ATPase in tissues varies greatly; a large difference exists between the lowest (i.e., 250–500 sites/cell in erythrocytes) and the highest (i.e., 11,000–12,000 pmol/g wet weight in the brain cortex) concentrations. The Na+/K+-ATPase could be pharmacologically modified by administrating drugs exogenously. For instance, Na+/K+-ATPase found in membranes of heart cells is an important target of cardiac glycosides (CG), inotropic drugs used to improve heart performance by increasing its force of contraction (Therien and Blostein 2000; Kaplan 2002) (Fig. 1).
Function and Regulation of Na+/K+-ATPase
Hormones and environmental factors influence Na+/K+-ATPase function through different mechanisms including regulation of activity, enzyme trafficking, gene expression, and phosphorylation (Therien and Blostein 2000; Al-Khalili et al. 2004; Sudar et al. 2008; Obradovic et al. 2014). Control and regulation of Na+/K+-ATPase activities via a direct influence on the kinetic properties of the enzyme that is already present in the plasma membrane occur within minutes to hours. The second mechanism of regulation means that new Na+/K+-ATPase subunits are delivered to the plasma membrane from intracellular stores (underneath the plasma membrane) when needed. Regulation of Na+/K+-ATPase subunit gene expression happens over days, while changes in the turnover rate of the existing Na+/K+-ATPase through its direct phosphorylation are still controversial. The main hormones that regulate the Na+/K+-ATPase in the above-mentioned ways are: aldosterone, insulin, androgen, thyroid hormone, and estrogen.
Cardiac glycosides inhibit Na+/K+-ATPase activity and subsequently increase the concentration of intracellular Na+ ([Na+]i) (Bagrov et al. 2009). The concentration of [Na+]i is regulated by balance of Na+ influx and efflux mechanisms, and the Na+/K+-ATPase provides the only significant Na+ efflux pathway (Kaplan 2002). The inhibition of Na+/K+-ATPase by endogenous CG in myocytes leads to an increased Na+ concentration, followed by increased [Ca2+]i (via NCX). This increase in [Ca2+]i content triggers the release of Ca2+ from the sarcoplasmatic reticulum, resulting in increased heart contraction. The Na+/K+-ATPase also acts as a signal transducer activating a number of intracellular pathways. Different molecules including CG or hormones binding to the extracellular region of the α-subunit activate signaling cascade and regulate different processes (Aperia 2007); unfortunately, this mechanism is still poorly understood. It has been reported that noninhibitory doses of ouabain triggered the Na+/K+-ATPase-dependent activation of the inositol 1,4,5-trisphosphate receptor (IP3R) via a direct interaction, resulting in increase of oscillatory [Ca2+]i content (Aperia 2007). Ca2+ oscillations appeared as the most versatile of all Ca2+ oscillations generated by the Na+/K+-ATPase–IP3R complex having a pleiotropic action on cell signals (Aperia 2007).
Short-term regulation of Na+/K+-ATPase activity includes changes in kinetic behavior of the Na+/K+-ATPase that already exist in the membrane (Therien and Blostein 2000). This mechanism of Na+/K+-ATPase regulation occurs within minutes to hours in response to changes in ion concentration, CG binding, and hormones action via PKA, PKC, Akt, PKG, or extracellular signal-regulated kinases 1 and 2 (Therien and Blostein 2000; Al-Khalili et al. 2004; Sudar et al. 2008; Obradovic et al. 2013). Although evidence suggests that phosphorylation/dephosphorylation of the Na+/K+-ATPase α-subunit mediated by protein kinases leads to changes in transport properties, this mechanism of regulation is still controversial (Therien and Blostein 2000; Fuller et al. 2013). Long-term regulation of Na+/K+-ATPase includes regulation of translocation of new Na+/K+-ATPase subunits from intracellular stores to the plasma membrane and transcription regulation of α- and β-subunit genes and de novo synthesis.
Environmental factors such as Na+ and K+ ions, ATP, and CG for which α-subunit of Na+/K+-ATPase contains the binding sites are responsible for instant regulation of Na+/K+-ATPase activity. Major determinant of Na+/K+-ATPase activity is the level of [Na+]i, where [Na+]i of 70–100 mM causes maximal activity. On the other hand, increased intracellular K+ ([K+]i) reduces Na+/K+-ATPase activity because of its competition with [Na+]i for binding to the E1 form. During ion pumping cycle, the Na+/K+-ATPase undergoes large-scale domain rearrangements while transitioning between two conformational states, E1 and E2, and sufficient concentration of ATP is necessary to allow this process (Shinoda et al. 2009). Endogenous CG (also known as digoxin-like immunoreactive factors) are produced in mammals in a manner similar to the production of steroid hormones and are involved in the regulation of inotropy effect and blood pressure (Bagrov et al. 2009). In kidneys, CG bind to α-subunit and subsequently inhibit renal tubular Na+/K+-ATPase, which causes natriuresis and diuresis. However, CG binding to the Na+/K+-ATPase induces generation of reactive oxygen species (ROS) in heart and kidneys. This increase of ROS can induce Na+/K+-ATPase oxidation and its conformational changes and inhibition of activity, while ultimately may cause its degradation. The impact of ROS on Na+/K+-ATPase is particularly significant in several diseases which are characterized by the elevation of endogenous ROS level, including heart failure. Glutathionylation, reversible oxidative modification of the β1 subunit of Na+/K+-ATPase, is another important mechanism of its regulation (Figtree et al. 2009). Additionally, glutathionylation of FXYD proteins is critical for reversal of β1 subunit glutathionylation and Na+/K+-ATPase inhibition induced by exposure of myocytes to angiotensin II and chemical oxidants. Also, growing evidence suggests that binding of ouabain to the Na+/K+-ATPase in noninhibitory doses may initiate signal transduction and modulate cell proliferation, apoptotic threshold, cell to cell contact, and cell migration. One more and relatively new type of Na+/K+-ATPase regulation involves regulation of associated FXYD proteins (Garty and Karlish 2006). Phospholemman (PLM, FXYD1) is a protein responsible for regulation of the cardiac Na+/K+-ATPase. PLM contains PKA and PKC phosphorylation sites on its cytoplasmic C-terminal tail and responds to adrenergic and other hormonal signals. Unphosphorylated PLM tonically inhibits Na+/K+-ATPase by decreasing the affinity for [Na+]i, and this inhibition is relieved by PKA-mediated phosphorylation of PLM at Ser68 and/or PKC-mediated phosphorylation of PLM at Ser63, Ser68, or Thr69 residues (Fuller et al. 2009). Stimulation of β1/β2-adrenergic receptors increases the level of cyclic adenosine-monophosphate (cAMP) and induces activation of PKA, which phosphorylates PLM at Ser68. Activation of PKA and, consequently, stimulation of the Na+/K+-ATPase via Ser68 PLM phosphorylation, limits [Na+]i and [Ca2+]i by favoring Ca2+ extrusion via NCX exchanger. Increased [Na+]i in the myocardium in heart failure favors more Ca2+ influx (through NCX) and better contractility in failing hearts. However, a chronic increase in [Na+]i and [Ca2+]i levels is also associated with dysfunctional cardiac hypertrophy and arrhythmogenesis. Stimulation of the Na+/K+-ATPase mediated by PLM phosphorylation may protect from Ca2+ overload, reducing the likelihood for triggered arrhythmias. In addition, phosphorylation of PLM Ser68 and cardiac Na+/K+-ATPase activity are negatively regulated by protein phosphatase 1 (PP1), which has been implicated in the regulation of cardiac β-agonist responses and contractility (El-Armouche et al. 2011). In failing human hearts, β-AR signaling is impaired, PLM Ser68 phosphorylation diminished, and PP1 potentially hyperactivated. CG are produced in mammals in a manner similar to steroid hormones from cholesterol and act as indirect regulators of cardiac contractility (positive inotropy) (Lingrel 2010).
Reduced Na+/K+-ATPase function seems to play a causal role in the development of cardiovascular (CV) diseases, probably due to the association of decreased Na+/K+-ATPase activity with other risk factors (e.g., obesity or impaired estradiol signaling) (Schwinger et al. 2003; Obradovic et al. 2013; Borović et al. 2016). Thus, the regulation of Na+/K+-ATPase activity and expression as well as the regulation of different Na+/K+-ATPase isoforms may be important for the treatment and possible prevention of CV diseases.
The Na+/K+-ATPase is a universally expressed membrane protein responsible for maintaining the low intracellular Na+ and high intracellular K+ concentrations required for multitude of cellular functions. Besides, the Na+/K+-ATPase helps maintaining resting potential, import of amino acids, glucose, and other nutrients into cells and regulates cellular volume. This enzyme has oligomeric structure consisting of two main subunits α and β and one auxiliary subunit FXYD. Subunits α and β are present in equal molar ratio and they combine to form a number of Na+/K+-ATPase isozymes (1:1), while FXYD proteins are present in cell and tissue-specific manner. Numerous hormones and environmental factors regulate Na+/K+-ATPase through several ways: by influencing Na+/K+-ATPase activity, changing subunit gene expression, regulating enzyme trafficking to the plasma membrane, and via phosphorylation of Na+/K+-ATPase which are present on membrane. The Na+/K+-ATPase found in the membrane of cardiomyocytes is an important target of CG, which improve heart performance by increasing contraction. Reduction of Na+/K+-ATPase activity/expression is associated with different pathophysiological conditions. Thus, increasing our understanding of the molecular mechanisms determining the regulation of Na+/K+-ATPase may help develop new strategies for the treatment of CV diseases.
This work is supported by grants No.173033 (to E.R.I) from the Ministry of Science, Republic of Serbia.
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