Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

Route,mechanism, and implications of proton import during Na+/K+ exchange by native Na+/K+-ATPase pumps

A single Na⁺/K⁺-ATPase pumps three Na⁺ outwards and two K⁺ inwards by alternately exposing ion-binding sites to opposite sides of the membrane in a conformational sequence coupled to pump autophosphorylation from ATP and auto-dephosphorylation. The larger flow of Na⁺ than K⁺ generates outward current across the cell membrane. Less well understood is the ability of Na⁺/K⁺ pumps to generate an inward current of protons. Originally noted in pumps deprived of external K⁺ and Na⁺ ions, as inward current at negative membrane potentials that becomes amplified when external pH is lowered, this proton current is generally viewed as an artifact of those unnatural conditions. We demonstrate here that this inward current also flows at physiological K⁺ and Na⁺ concentrations. We show that protons exploit ready reversibility of conformational changes associated with extracellular Na⁺ release from phosphorylated Na⁺/K⁺ pumps. Reversal of a subset of these transitions allows an extracellular proton to bind an acidic side chain and to be subsequently released to the cytoplasm. This back-step of phosphorylated Na⁺/K⁺ pumps that enables proton import is not required for completion of the 3 Na⁺/2 K⁺ transport cycle. However, the back-step occurs readily during Na⁺/K⁺ transport when external K⁺ ion binding and occlusion are delayed, and it occurs more frequently when lowered extracellular pH raises the probability of protonation of the externally accessible carboxylate side chain. The proton route passes through the Na⁺-selective binding site III and is distinct from the principal pathway traversed by the majority of transported Na⁺ and K⁺ ions that passes through binding site II. The inferred occurrence of Na⁺/K⁺ exchange and H⁺ import during the same conformational cycle of a single molecule identifies the Na⁺/K⁺ pump as a hybrid transporter. Whether Na⁺/K⁺ pump–mediated proton inflow may have any physiological or pathophysiological significance remains to be clarified.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.     

周期性张应变对面颌部肌细胞Na+/K+-ATPaseα亚单位蛋白表达的影响

目的:研究周期性张应变对面颌肌细胞Na+/K+-ATPaseα亚单位蛋白表达的影响以确定其作用及机制.方法:在建立面颌肌细胞的力学刺激-细胞体外培养模型的基础上,采用Western blot法分析周期性张应变对Na+/K+-ATPase α1和α2亚单位蛋白表达的影响,加力组分别给予1、2、12、24和48h的力学刺激,施加力值为15％的细胞形变,频率为10cycles/min.以静态组为对照组.对照组及实验组各包含4个实验样本.Western blot检测Na+/K+-ATPaseα1和α2亚单位蛋白的表达.结果:α1亚单位的蛋白表达量除加力1h组与对照组之间、加力24 h与48 h之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义.α2亚单位蛋白表达量除加力24 h与48 h组之间无统计学差异外,其余各组之间以及各组与对照组之间均有显著的统计学意义.结论:在一定时间范围内,周期性张应变可刺激α1和α2亚单位蛋白表达增加,随作用时间的延长蛋白表达受抑制.提示在肌能力的刺激下,面颌肌细胞的相关酶蛋白的功能及表达将发生适应性改建,但其功能亚基的调控机制可能不同.这为选择不同的方法和手段进行临床干预提供了理论依据,因而具有重要的参考意义.     

Na+,K+-ATPase Na+ Affinity in Rat Skeletal Muscle Fiber Types

Previous studies in expression systems have found different ion activation of the Na⁺/K⁺-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na⁺,K⁺-ATPase activity, and the Na⁺ affinity of Na⁺,K⁺-ATPase was studied in total membranes from rat muscle and purified membranes from muscle with different fiber types. The Na⁺ affinity was higher (K _m lower) in oxidative muscle compared with glycolytic muscle and in purified membranes from oxidative muscle compared with glycolytic muscle. Na⁺,K⁺-ATPase isoform analysis implied that heterodimers containing the β₁ isoform have a higher Na⁺ affinity than heterodimers containing the β₂ isoform. Immunoprecipitation experiments demonstrated that dimers with α₁ are responsible for approximately 36% of the total Na,K-ATPase activity. Selective inhibition of the α₂ isoform with ouabain suggested that heterodimers containing the α₁ isoform have a higher Na⁺ affinity than heterodimers containing the α₂ isoform. The estimated K _m values for Na⁺ are 4.0, 5.5, 7.5 and 13 mM for α₁β₁, α₂β₁, α₁β₂ and α₂β₂, respectively. The affinity differences and isoform distributions imply that the degree of activation of Na⁺,K⁺-ATPase at physiological Na⁺ concentrations differs between muscles (oxidative and glycolytic) and between subcellular membrane domains with different isoform compositions. These differences may have consequences for ion balance across the muscle membrane.     

Gramicidin A directly inhibits mammalian Na+/K+-ATPase

The linear pentadecapeptide gramicidin A forms an ion channel in the lipid bilayer to selectively transport monovalent cations. Nevertheless, we have surprisingly found that gramicidin A directly inhibits mammalian Na(+)/K(+)-ATPase. Gramicidin A inhibited ATP hydrolysis by Na(+)/K(+)-ATPase from porcine cerebral cortex at the IC(50) value of 8.1 microM, while gramicidin S was approximately fivefold less active. The synthetic gramicidin A analog lacking N-terminal formylation and C-terminal ethanolamine exhibited a weaker inhibitory effect on the ATP-hydrolyzing activity of Na(+)/K(+)-ATPase than gramicidin A, indicating that these end modifications are necessary for gramicidin A to inhibit Na(+)/K(+)-ATPase activity. Moreover, Lineweaver-Burk analysis showed that gramicidin A exhibits a mixed type of inhibition. In addition to the most well-studied ionophore activity, our present study has disclosed a novel biological function of gramicidin A as a direct inhibitor of mammalian Na(+)/K(+)-ATPase activity.     

Eosin fluorescence changes during Rb+ occlusion in the Na+/K(+)-ATPase

We used suspensions of partially purified Na(+)/K(+)-ATPase from pig kidney to compare the effects of Rb(+), as a K(+) congener, on the time course and on the equilibrium values of eosin fluorescence and of Rb(+) occlusion. Both sets of data were collected under identical conditions in the same enzyme preparations. The incubation media lacked ATP so that all changes led to an equilibrium distribution between enzyme conformers with and without bound eosin and with and without bound or occluded Rb(+). Results showed that as Rb(+) concentration was increased, the equilibrium value of fluorescence decreased and occlusion increased along rectangular hyperbolas with similar half-maximal values. The time courses of attainment of equilibrium showed an initial phase which was so quick as to fall below the time resolution of our rapid-mixing apparatus. This phase was followed by the sum of at least two exponential functions of time. In the case of fluorescence the fast exponential term accounted for a larger fraction of the time course than in the case of occlusion. Comparison between experimental and simulated results suggests that fluorescence changes express a process that is coupled to Rb(+) occlusion but that is completed before occlusion reaches equilibrium.     

Activation of the Na+/K+-ATPase by interleukin-2

Activated B61.SF.1 and CTLL-2 T lymphocyte clones which are strictly dependent on interleukin-2 (IL-2) for growth were used to study the activation of Na+/K+-ATPase. 50% of [3H]thymidine maximal incorporation was obtained when the extracellular concentration of Na+ or K+ was reduced to 50 or 2 mM, respectively. 'Quiescent' CTL clones stimulated with IL-2 showed an increase of 48-380% in ouabain-sensitive 86Rb uptake. Furthermore, this stimulation was completely inhibited by a monoclonal antibody PC.61 directed at the IL-2 receptor. The activation of the pump was dependent on the dose of IL-2, took place at the same doses of IL-2 that were required to stimulate cell proliferation and was linear for at least 30 min.     

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.     

Ethylenediamine as active site probe for Na+/K+-ATPase

(1) Ethylenediamine is an inhibitor of Na+- and K+-activated processes of Na+/K+-ATPase, i.e. the overall Na+/K+-ATPase activity, Na+-activated ATPase and K+-activated phosphatase activity, the Na+-activated phosphorylation and the Na+-free (amino-buffer associated) phosphorylation. (2) The I50 values (I50 is the concentration of inhibitor that half-maximally inhibits) increase with the concentration of the activating cations and the half-maximally activating cation concentrations (Km values) increase with the inhibitor concentration. (3) Ethylenediamine is competitive with Na+ in Na+-activated phosphorylation and with the amino-buffer (triallylamine) in Na+-free phosphorylation. Significant, though probably indirect, effects can also be noted on the affinity for Mg2+ and ATP, but these cannot account for the inhibition. (4) Inhibition parallels the dual protonated or positively charged ethylenediamine concentration (charge distance 3.7 A). (5) Direct investigation of interaction with activating cations (Na+, K+, Mg+, triallylamine) has been made via binding studies. All these cations drive ethylenediamine from the enzyme, but K+ and Mg+ with the highest efficiency and specificity. Ethylenediamine binding is ouabain-insensitive, however. (6) Ethylenediamine neither inhibits the transition to the phosphorylation enzyme conformation, nor does it affect the rate of dephosphorylation. Hence, we provisionally conclude that ethylenediamine inhibits the phosphoryl transfer between the ATP binding and phosphorylation site through occupation of cation activation sites, which are 3-4 A apart.     

The pathway for spontaneous occlusion of Rb+ in the Na+/K+-ATPase

Occlusion of K (+) in the Na (+)/K (+)-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na (+) and Mg (2+), after the K (+)-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K (+) to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb (+) in an unsided, purified preparation of Na (+)/K (+)-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K (+) and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg (2+) plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg (2+) plus Pi, exchange of K (+) would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb (+) ions was labeled as (86)Rb (+). We found that, when the enzyme is in the E2 conformation, the first Rb (+) that entered the enzyme in media without Mg (2+) and Pi was the last to leave after addition of Mg (2+) plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb (+) between E2(Rb 2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K (+) during the ATPase activity.     

Binding of manganese ions to the Na+/K+-ATPase during phosphorylation by ATP

The aim of the present work was to study the Mg2+-Na+/K+-ATPase interaction that was proposed to lead to the formation of a stable Mg-enzyme complex during phosphorylation from ATP. Instead of Mg we used Mn, which can replace Mg as essential activator of Na+/K+-ATPase activity. The amounts of steady-state Mn bound to the enzyme were estimated at 0 degree C on the basis of the 54Mn remaining in the effluent after passing the reaction mixture through a cation exchange resin column. As a function of the MnCl2 concentration, the amount of Mn retained by the enzyme in the absence and presence of ATP showed a saturable and a linear component; the slope of the linear component was the same in both instances (0.016 nmol/mg per microM). The ATP-dependent Mn binding could be adjusted to a hyperbolic function with a Km of 0.76 microM. The ratio [ATP-dependent E-Mn]/[E-P] measured at 5 microM MnCl2 and 5 microM ATP was not different from 1.0, both in native (Mn-E2-P) as well as in a chymotrypsin treated enzyme (Mn-E1-P). When the Mn.E-P complex was allowed to react with KCl (E2-P form) or ADP (E1-P form), the enzyme was dephosphorylated and simultaneously lost the strongly bound Mn in such a way that the ratio [ATP-dependent E-Mn]/[E-P] remained 1:1. These results show the existence of strongly bound Mn ions to Na+/K+-ATPase during phosphorylation by ATP. That binding is (i) of high affinity for Mn, (ii) probably on a single site, and (iii) with a stoichiometry Mn-Pi of 1:1.     

Na+/K+ -ATPase regulates tight junction formation and function during mouse preimplantation development

Research applied to the early embryo is required to effectively treat human infertility and to understand the primary mechanisms controlling development to the blastocyst stage. The present study investigated whether the Na(+)/K(+)-ATPase regulates tight junction formation and function during blastocyst formation. To investigate this hypothesis, three experimental series were conducted. The first experiments defined the optimal dose and treatment time intervals for ouabain (a potent and specific inhibitor of the Na(+)/K(+)-ATPase) treatment. The results demonstrated that mouse embryos maintained a normal development to the blastocyst stage following a 6-h ouabain treatment. The second experiments investigated the effects of ouabain treatment on the distribution of ZO-1 and occludin (tight junction associated proteins). Ouabain treatment (up to 6 h) or culture in K(+)-free medium (up to 6 h) resulted in the appearance of a discontinuous ZO-1 protein distribution and a loss of occludin immunofluorescence. The third set of experiments examined the influence of ouabain treatment on tight junction function. Ouabain treatment or culture in K(+)-free medium affected tight junction permeability as indicated by an increase in the proportion of treated embryos accumulating both 4 kDa and 40 kDa fluorescein isothiocyanate (FITC)-dextran into their blastocyst cavities. The results indicate that the Na(+)/K(+)-ATPase is a potent regulator of tight junction formation and function during mouse preimplantation development.