Mechanism of the Na,K-ATPase Inhibition by MCS Derivatives

The previously reported class of potent inorganic inhibitors of Na,K-ATPase, named MCS factors, was shown to inhibit not only Na,K-ATPase but several P-type ATPases with high potency in the sub-micromolar range. These MCS factors were found to bind to the intracellular side of the Na, K-ATPase. The inhibition is not competitive with ouabain binding, thus excluding its role as cardiac-steroid-like inhibitor of the Na,K-ATPase. The mechanism of inhibition of Na,K-ATPase was investigated with the fluorescent styryl dye RH421, a dye known to report changes of local electric fields in the membrane dielectric. MCS factors interact with the Na,K-ATPase in the E₁ conformation of the ion pump and induce a conformational rearrangement that causes a change of the equilibrium dissociation constant for one of the first two intracellular cation binding sites. The MCS-inhibited state was found to have bound one cation (H⁺, Na⁺ or K⁺) in one of the two unspecific binding sites, and at high Na⁺ concentrations another Na⁺ ion was bound to the highly Na⁺-selective ion-binding site.     

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na⁺/K⁺ ion binding site of the Na,K-ATPase α subunit.     

Polarized membrane distribution of potassium-dependent ion pumps in epithelial cells: Different roles of the <Emphasis Type="Italic">N</Emphasis>-glycans of their <Emphasis Type="Italic">β</Emphasis> subunits

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P₂-type pumps that catalyze active transport of Na⁺ in exchange for K⁺ (Na,K-ATPase) or H⁺ in exchange for K⁺ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β ₁-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β ₁-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β ₁-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β ₁–β ₁ interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β ₂-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β ₂-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit. Supported in part by NIH grants DK46917, DK58333, D53642, and USVA     

The Role of Oxidized Nicotinamide Adenine Dinucleotide in Fluoride Inhibition of Active Sodium Transport in Human Erythrocytes

The rate coefficient for ²²Na release from previously labeled human erythrocytes was determined in the presence of 0.1–10 mM sodium fluoride (F). The oxidized nicotinamide adenine dinucleotide (NAD⁺) level at the end of 2 hr of incubation in tris(hydroxymethyl)aminomethane (Tris)-Ringer medium was also measured. Both parameters decreased proportionately as F concentration was raised. Both F-induced changes were immediate and were reversed by 10 mM pyruvate. The decrease in NAD⁺ concentration following enolase inhibition by F is attributed to a diminished rate of formation in the reaction catalyzed by lactic dehydrogenase (LDH) with undiminished continued utilization in the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). It is postulated that the NAD⁺ lowering limited the GAPDH step, resulting in proportionate decreases in the rates of phosphoglycerate kinase (PGK) and Na,K-dependent adenosine triphosphatase (Na,K-ATPase), a reaction sequence thought to link glycolysis with active Na extrusion. Adding pyruvate with F increased NAD⁺ production at the LDH step, thus reactivating GAPDH, PGK, and Na,K-ATPase and leading to the observed restoration of ²²Na release. The results suggest, therefore, that F inhibits active Na transport in intact human erythrocytes indirectly through a lowering of NAD⁺, although, direct inhibition of the Na,K-ATPase by F may possibly occur simultaneously.     

A Link between FXYD3 (Mat-8)-mediated Na,K-ATPase Regulation and Differentiation of Caco-2 Intestinal Epithelial Cells

FXYD3 (Mat-8) proteins are regulators of Na,K-ATPase. In normal tissue, FXYD3 is mainly expressed in stomach and colon, but it is also overexpressed in cancer cells, suggesting a role in tumorogenesis. We show that FXYD3 silencing has no effect on cell proliferation but promotes cell apoptosis and prevents cell differentiation of human colon adenocarcinoma cells (Caco-2), which is reflected by a reduction in alkaline phosphatase and villin expression, a change in several other differentiation markers, and a decrease in transepithelial resistance. Inhibition of cell differentiation in FXYD3-deficient cells is accompanied by an increase in the apparent Na⁺ and K⁺ affinities of Na,K-ATPase, reflecting the absence of Na,K-pump regulation by FXYD3. In addition, we observe a decrease in the maximal Na,K-ATPase activity due to a decrease in its turnover number, which correlates with a change in Na,K-ATPase isozyme expression that is characteristic of cancer cells. Overall, our results suggest an important role of FXYD3 in cell differentiation of Caco-2 cells. One possibility is that FXYD3 silencing prevents proper regulation of Na,K-ATPase, which leads to perturbation of cellular Na⁺ and K⁺ homeostasis and changes in the expression of Na,K-ATPase isozymes, whose functional properties are incompatible with Caco-2 cell differentiation.     

The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter trafficking

The Na/K-ATPase was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind a group of chemicals called cardiotonic steroids (CTS). The plant-derived CTS such as digoxin are valuable drugs for the management of cardiac diseases, whereas ouabain and marinobufagenin (MBG) have been identified as a new class of endogenous hormones. Recent studies have demonstrated that the endogenous CTS are important regulators of renal Na⁺ excretion and blood pressure. The Na/K-ATPase is not only an ion pump, but also an important receptor that can transduce the ligand-like effect of CTS on intracellular protein kinases and Ca²⁺ signaling. Significantly, these CTS-provoked signaling events are capable of reducing the surface expression of apical NHE3 (Na/H exchanger isoform 3) and basolateral Na/K-ATPase in renal proximal tubular cells. These findings suggest that endogenous CTS may play an important role in regulation of tubular Na⁺ excretion under physiological conditions; conversely, a defect at either the receptor level (Na/K-ATPase) or receptor–effector coupling would reduce the ability of renal proximal tubular cells to excrete Na⁺, thus culminating/resulting in salt-sensitive hypertension.     

Effects of γ-irradiation on Na,K-ATPase in cardiac sarcolemma

Previous studies showed that adverse effect of ionizing radiation on the cardiovascular system is beside other factors mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. One of the structures highly sensitive to radicals is the Na,K-ATPase the main system responsible for extrusion of superfluous Na⁺ out of the cell which utilizes the energy derived from ATP. The aim of present study was the investigation of functional properties of cardiac Na,K-ATPase in 20-week-old male rats 6 weeks after γ-irradiation by a dose 25 Gy (IR). Irradiation induced decrease of systolic blood pressure from 133 in controls to 85 mmHg in IR group together with hypertrophy of right ventricle (RV) and hypotrophy of left ventricle (LV). When activating the cardiac Na,K-ATPase with substrate, its activity was lower in IR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (V _max) by 40 % with no changes in the value of Michaelis–Menten constant (K _m). During activation with Na⁺, we observed a decrease of the enzyme activity in hearts from IR at all tested Na⁺ concentrations. The value of V _max decreased by 38 %, and the concentration of Na⁺ that gives half maximal reaction velocity (K _Na) increased by 62 %. This impairment in the affinity of the Na⁺-binding site together with decreased number of active Na,K-ATPase molecules, as indicated by lowered V _max values, are probably responsible for the deteriorated efflux of the excessive Na⁺ from the intracellular space in hearts of irradiated rats.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the β₁ subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH₄⁺ as activating cation. The enzyme was also stimulated by Na⁺, but in contrast to the rat enzyme, hardly by K⁺. SCH 28080 inhibited the NH₄⁺-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K⁺], and [Na⁺] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E₁P to E₂P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H⁺/Na⁺ and/or H⁺/NH₄⁺ transport but not in Na⁺/K⁺ transport.     

Function of FXYD Proteins,Regulators of Na,K-ATPase

In this short review, we summarize our work on the role of members of the FXYD protein family as tissue-specific modulators of Na, K-ATPase. FXYD1 or phospholemman, mainly expressed in heart and skeletal muscle increases the apparent affinity for intracellular Na⁺ of Na, K-ATPase and may thus be important for appropriate muscle contractility. FXYD2 or γ subunit and FXYD4 or CHIF modulate the apparent affinity for Na⁺ of Na, K-ATPase in an opposite way, adapted to the physiological needs of Na⁺ reabsorption in different segments of the renal tubule. FXYD3 expressed in stomach, colon, and numerous tumors also modulates the transport properties of Na, K-ATPase but it has a lower specificity of association than other FXYD proteins and an unusual membrane topology. Finally, FXYD7 is exclusively expressed in the brain and decreases the apparent affinity for extracellular K⁺, which may be essential for proper neuronal excitability.     

Osmotic stress and viscous retardation of the Na,K-ATPase ion pump

The transport function of the Na pump (Na,K-ATPase) in cellular ion homeostasis involves both nucleotide binding reactions in the cytoplasm and alternating aqueous exposure of inward- and outward-facing ion binding sites. An osmotically active, nonpenetrating polymer (poly(ethyleneglycol); PEG) and a modifier of the aqueous viscosity (glycerol) were used to probe the overall and partial enzymatic reactions of membranous Na,K-ATPase from shark salt glands. Both inhibit the steady-state Na,K-ATPase as well as Na-ATPase activity, whereas the K⁺-dependent phosphatase activity is little affected by up to 50% of either. Both Na,K-ATPase and Na-ATPase activities are inversely proportional to the viscosity of glycerol solutions in which the membranes are suspended, in accordance with Kramers’ theory for strong coupling of fluctuations at the active site to solvent mobility in the aqueous environment. PEG decreases the affinity for Tl⁺ (a congener for K⁺), whereas glycerol increases that for the nucleotides ATP and ADP in the presence of NaCl but has little effect on the affinity for Tl⁺. From the dependence on osmotic stress induced by PEG, the aqueous activation volume for the Na,K-ATPase reaction is estimated to be ∼5-6 nm³ (i.e., ∼180 water molecules), approximately half this for Na-ATPase, and essentially zero for p-nitrophenol phosphatase. The change in aqueous hydrated volume associated with the binding of Tl⁺ is in the region of 9 nm³. Analysis of 15 crystal structures of the homologous Ca-ATPase reveals an increase in PEG-inaccessible water space of ∼22 nm³ between the E₁-nucleotide bound forms and the E₂-thapsigargin forms, showing that the experimental activation volumes for Na,K-ATPase are of a magnitude comparable to the overall change in hydration between the major E₁ and E₂ conformations of the Ca-ATPase.     

Some characteristics of Na/K-ATPase from rat intestinal basal lateral membranes

Summary Basal lateral membrane vesicles were isolated from rat intestinal epithelial cells. The sodium potassium triphosphatase (Na/K-ATPase) of these plasma membranes has been characterized by (1) the molecular weight of the phosphorylated intermediate, (2) the sensitivity of the phosphorylated intermediate to hydroxylamine, (3) its ouabain binding constants, and (4) its susceptibility to digestion by pronase. The phosphorylated intermediate was shown by SDS polyacrylamide gel electrophoresis to be a protein of 100,000 Daltons apparent mol wt. Its extensive hydrolysis in hydroxylamine demonstrated that it was an acyl phosphate. The isolated basal lateral membranes bound ouabain with a dissociation constant,K _m (1.5×10^–5 m), similar to the inhibitory constantK _I (3×10^–5 m), measured for ouabain inhibition of the Na/K-ATPase activity. The association rate constant measured for ouabain binding at 22°C was 1.3×10³ m ^–1 sec^–1 and is similar to the association rate constants reported for other tissues and species. The high dissociation rate constant, 3.6×10^–2 sec^–1, is consistent with the insensitivity of the rat to ouabain. Digestion of the intact cells by pronase yielded basal lateral membranes in which the Na/K-ATPase had been unaffected. The phosphorylated intermediate ran as a sharp band at 100,000 Daltons on electrophoresis, and the ouabain dissociation constant appeared to be unchanged. In these membranes, protein stains of polyacrylamide gels revealed digestion of the major high mol wt proteins including the major protein at 100,000 Daltons. This suggests that the Na/K-ATPase represent a minor component, less than 1%, of the basal lateral membrane protein. From these characteristics of the phosphorylated intermediate and the ouabain binding constants, we conclude that the Na/K-ATPase of the basal lateral membranes of rat intestinal epithelial cells is similar to that found in other tissues and species. Estimates of the number of pump sites and the turnover number predict rates of Na transport that are consistent with observed values.This paper is dedicated to the memory of Professor David H. Smyth, FRS, who died on September 10, 1979.     

Effects of Ganoderma Lucidum shell-broken spore on oxidative stress of the rabbit urinary bladder using an in vivo model of ischemia/reperfusion

The present study was oriented to gender specificity of Na,K-ATPase in cerebellum, the crucial enzyme maintaining the intracellular homeostasis of Na ions in healthy and diabetic Wistar rats. The effects of diabetes on properties of the Na,K-ATPase in cerebellum derived from normal and streptozotocin (STZ)-diabetic rats of both genders were investigated. The samples were excised at different time intervals of diabetes induced by STZ (65 mg kg^?1) for 8 days and 16 weeks. In acute 8-day-lasting model of diabetes, Western blot analysis showed significant depression of α1 isoform of Na,K-ATPase in males only. On the other hand, concerning the activity, the enzyme seems to be resistant to the acute model of diabetes in both genders. Prolongation of diabetes to 16 weeks was followed by increasing the number of active molecules of Na,K-ATPase exclusively in females as indicated by enzyme kinetic studies. Gender specificity was observed also in nondiabetic animals revealing higher Na,K-ATPase activity in control males probably caused by higher number of active enzyme molecules as indicated by increased value of V _max when comparing to control female group. This difference seems to be age dependent: at the age of 16 weeks, the V _max value in females was higher by more than 90%, whereas at the age of 24 weeks, this difference amounted to only 28%. These data indicate that the properties of Na,K-ATPase in cerebellum, playing crucial role in maintaining the Na⁺ and K⁺ gradients, depend on gender, age, and duration of diabetic impact.     

Stimulation of Na,K-ATPase by Low Potassium Is Dependent on Transferrin

We took advantage of the fact that confluent MDCK cells can survive in a serum-free medium for several days to examine whether the upregulation of Na,K-ATPase by low K⁺ required serum. We found that serum was essential for low K⁺ to induce an increase in the cell surface Na,K-ATPase molecular number as quantified by ouabain binding assays. Further analyses identified that transferrin, not EGF or IGF-1, could simulate the effect of serum. Moreover, transferrin was also required for low-K⁺-induced increases in 1-subunit promoter activity, 1- and 1-subunit protein abundance of the Na,K-ATPase. In the presence of transferrin, low K⁺ enhanced cellular uptake of iron. Inhibition of intracellular iron activity by deferoxamine (40 µM) abrogated the effect of low K⁺ on the Na,K-ATPase. Like deferoxamine, catalase (100 U/ml) also ablated the effect of low K⁺. We conclude that stimulation of the Na,K-ATPase by low K⁺ is dependent on transferrin. The effect of transferrin is mediated by increased iron transport and reactive oxygen species activity.     

Isoform-specific functions of Na,K-ATPase in skeletal muscle

The published data and the results of the author’s own research in the field of the molecular and functional diversity of Na,K-ATPases are reviewed. Na,K-ATPase is an integral membrane protein that maintains the concentration gradients of Na⁺ and K⁺ that are essential for electrogenesis, excitability, and several other processes of cellular transport. Most of the Na,K-ATPase of vertebrates is found in the skeletal muscle tissue, which co-expresses the α1 and α2 isoforms of the catalytic and transport α-subunit of Na,KATPase. The activity of Na,K-ATPase is crucial for the contractile function and prolonged activity of skeletal muscle. The data that have accumulated indicate that the α1 isoform of Na,K-ATPase fulfills the major pumping function. The α2 isoform fulfills additional functions related to the specific membrane localization of the protein, the functional interactions with the proteins and lipids of the environment, and fine-tuned regulation by a variety of factors, including motor activity.     

Na,K-ATPase structure/function relationships probed by the denaturant urea

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37 °C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K⁺ as well as the K⁺-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na⁺. Half-maximal inhibition is seen with about 1 M urea for shark enzyme and about 2 M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation.     

Na,K-ATPase α2 activity in mammalian skeletal muscle T-tubules is acutely stimulated by extracellular K+

The Na,K-ATPase α2 isoform is the predominant Na,K-ATPase in adult skeletal muscle and the sole Na,K-ATPase in the transverse tubules (T-tubules). In quiescent muscles, the α2 isozyme operates substantially below its maximal transport capacity. Unlike the α1 isoform, the α2 isoform is not required for maintaining resting ion gradients or the resting membrane potential, canonical roles of the Na,K-ATPase in most other cells. However, α2 activity is stimulated immediately upon the start of contraction and, in working muscles, its contribution is crucial to maintaining excitation and resisting fatigue. Here, we show that α2 activity is determined in part by the K⁺ concentration in the T-tubules, through its K⁺ substrate affinity. Apparent K⁺ affinity was determined from measurements of the K_1/2 for K⁺ activation of pump current in intact, voltage-clamped mouse flexor digitorum brevis muscle fibers. Pump current generated by the α2 Na,K-ATPase, Ip, was identified as the outward current activated by K⁺ and inhibited by micromolar ouabain. Ip was outward at all potentials studied (−90 to −30 mV) and increased with depolarization in the subthreshold range, −90 to −50 mV. The Q₁₀ was 2.1 over the range of 22–37°C. The K_1/2,K of Ip was 4.3 ± 0.3 mM at −90 mV and was relatively voltage independent. This K⁺ affinity is lower than that reported for other cell types but closely matches the dynamic range of extracellular K⁺ concentrations in the T-tubules. During muscle contraction, T-tubule luminal K⁺ increases in proportion to the frequency and duration of action potential firing. This K_1/2,K predicts a low fractional occupancy of K⁺ substrate sites at the resting extracellular K⁺ concentration, with occupancy increasing in proportion to the frequency of membrane excitation. The stimulation of preexisting pumps by greater K⁺ site occupancy thus provides a rapid mechanism for increasing α2 activity in working muscles.     

Investigation of mechanism of p38 MAPK activation in renal epithelial cell from distal tubules triggered by cardiotonic steroids

Ouabain and other cardiotonic steroids (CTS) kill renal epithelial cells from distal tubules (C7-MDCK) via interaction with Na,K-ATPase but independently of inhibition of Na,K-ATPase-mediated ion fluxes. Recently, we demonstrated that modest intracellular acidification and inhibition of p38 MAPK suppress death of C7-MDCK cells triggered by ouabain. In the present study we investigate the mechanism of p38 MAPK activation in renal epithelial cell from distal tubules evoked by cardiotonic steroids. Using Na⁺/K⁺ ionophores (monensin, nigericin) and media with different content of monovalent cations, we revealed that p38 MAPK phosphorylation in ouabain-treated renal epithelial cells is not caused by Na,K-ATPase inhibition and inversion of the [Na⁺]_i/[K⁺]_i ratio. We also demonstrated that attenuation of pH from 7.45 to 6.75 did not alter the level of p38 MAPK phosphorylation observed in ouabain-treated cells. Inhibitors of PKA, PKC, and PKG as well as protein phosphatases were unable to abolish p38 MAPK activation triggered by ouabain. Using phosphotyrosine antibodies we did not detect any effect of ouabain on activation of tyrosine kinases. Thus, our results show that activation of p38 MAPK and cytotoxic action of CTS are independent of intracellular Na⁺, K⁺, and H⁺ concentrations. The molecular origin of intermediates of death signaling induced by CTS via conformation changes of Na,K-ATPase with following activation of p38 MAPK should be examined further.     

Harmaline: A competitive inhibitor of Na Ion in the (Na++K+)-ATPase system

Summary Experimental evidence is given that the hallucinogen harmaline (HME) behaves as an inhibitor of the (Na⁺+K⁺)-ATPase system, specifically in the Na⁺-dependent phosphorylation reaction. HME at 0.3 to 3mm inhibited several membrane ATPase preparations such as those from human erythrocytes, rat brain and squid retinal axons. The same concentration blocked Na⁺ outflow from squid giant axons. The behavior of several harmane derivatives such as harmine, harmalol and harmaline demonstrated that certain groups influenced the concentration for 50% inhibition of the ATPase system. The following evidence demonstrated that HME blocked the formation of the phosphorylated intermediate by competition with Na ions in the (Na⁺+K⁺)-ATPase reaction in rat brain. (1) The HME effect on the overall (Na⁺+K⁺)-ATPase reaction showed a fully competitive inhibition with respect to Na ion concentration. (2) The inhibition of the Na⁺-stimulated phosphorylation by HME was fully competitive with respect to Na ions, with or without oligomycin present. (3) HME inhibited the effect of ADP on the phosphorylation reaction using³²P-ATP. (4) HME did not accelerate the rate of membrane dephosphorylation by means of³²P-ATP and cold ATP.From the behavior of HME as a competitive inhibitor at Na ion sites of the (Na⁺+K⁺)-ATPase reactions one may gain information about (a) The chemical nature of Na⁺ sites which may be responsible for the selectivity of this cation, and (b) The sequence of Na⁺ and ATP entrance into the Na⁺-dependent phosphorylation reaction. The experimental evidence supports the hypothesis that the entrance of Na⁺ into the enzyme system may precede the formation of the phosphorylated intermediate.