Cryptosin induces backbone structural changes in cardiac Na+ and K+ dependent adenosinetriphosphatase

Cryptosin, a new cardenolide, was found to preferentially bind to Na,K-ATPase enzyme (7), which is believed to be the ouabain binding site on cardiac sarcolemmal membrane. CD spectral studies revealed that cryptosin, in the presence of Na+ and Mg++ ions, bind to Na,K-ATPase and induce a dose-dependent change in the backbone structure of cardiac Na,K-ATPase.     

Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides

It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (I(P)) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of I(P) because of the following: (1) it was absent in 0 mM [K(+)](o), as was I(P); (2) it was absent in 0 mM [Na(+)](i), as was I(P); (3) at reduced [Na(+)](i), the outward current was reduced in proportion to the reduction in I(P); (4) it was eliminated by intracellular vanadate, as was I(P). Our previous work suggested guinea pig ventricular myocytes coexpress the alpha(1)- and alpha(2)-isoforms of the Na/K pumps. The stimulation of I(P) appears to be through stimulation of the high glycoside affinity alpha(2)-isoform and not the alpha(1)-isoform because of the following: (1) regulatory signals that specifically increased activity of the alpha(2)-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the alpha(1)-isoform did not affect the stimulation; (3) changes in [K(+)](o) that affected activity of the alpha(1)-isoform, but not the alpha(2)-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the alpha(1)-isoform but not the alpha(2)-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total I(P) increased by 35 +/- 10% (mean +/- SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the alpha(2)-isoform, then activity of the alpha(2)-isoform increased by 107 +/- 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the alpha(2)-isoform, but both the stimulatory and inhibitory concentrations of ouabain were approximately 10-fold lower than those for DHO. Stimulation of I(P) by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the alpha(1)- and alpha(3)-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the alpha(3)-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of I(P) that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of I(P), and where the contributions of the high glycoside affinity alpha(2)- and alpha(3)-isoforms could be separated from that of the alpha(1)-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of I(P) in heart by nanomolar concentrations of endogenous ouabain-like molecules.     

Calmodulin stimulates both adenosine 5'-triphosphate hydrolysis and synthesis catalyzed by a cardiac calcium ion dependent adenosinetriphosphatase

A Ca2+-dependent ATPase purified from a rabbit heart membrane preparation was compared to the Ca2+-dependent ATPase purified from skeletal muscle sarcoplasmic reticulum. The two ATPases display an identical electrophoretic pattern and an identical Ca2+-concentration dependence. However, only the cardiac preparation exhibits a 2-3-fold activation by calmodulin. This effect is best observed when the molar concentrations of calmodulin and ATPase are equivalent and in the presence of high Ca2+ (approximately 10(-5) M) and ATP (approximately 10(-3) M) concentrations. It is demonstrated for the first time that calmodulin stimulates the rate of ATP synthesis, as revealed by an increased production of Pi and a faster ATP in equilibrium Pi exchange, as well as the rate of ATP hydrolysis. It is also demonstrated that calmodulin activation is expressed with purified and detergent-solubilized enzyme in addition to membrane-bound systems. These findings indicate that the effect of calmodulin is an acceleration of the enzyme turnover, due to direct interaction of calmodulin with the enzyme.     

Reversible loss of cooperative calcium ion binding by sarcoplasmic reticulum calcium adenosinetriphosphatase

Incubation of rabbit skeletal muscle sarcoplasmic reticulum vesicles in solutions of very low [Ca2+] caused Ca2+ to bind noncooperatively, as determined by the dependence of the intrinsic tryptophan fluorescence intensity on added increments of Ca2+. Cooperative Ca2+ binding was obtained if the ATPase was incubated in [Ca2+] high enough (25 microM) to saturate the two high-affinity Ca2+ binding sites and then titrated with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The cooperative binding had an apparent association constant of 6.3 X 10(6) M-1 and a Hill coefficient of 2.6; these constants for the noncooperative binding case were 5.0 X 10(5) M-1 and 1.2, respectively. The transitions from the noncooperative to the cooperative Ca2+ binding forms of the enzyme were slow compared to the time required for Ca2+ binding to reach equilibrium. Thus, it appears that sarcoplasmic reticulum CaATPase is a hysteretic enzyme. Intrinsic association constants for Ca2+ binding and equilibrium constants for the transitions between the two forms in low and high [Ca2+] were estimated from analyses of a general scheme for cooperative and noncooperative binding.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Lipid selectivity of the calcium and magnesium ion dependent adenosinetriphosphatase, studied with fluorescence quenching by a brominated phospholipid

1,2-Bis(9,10-dibromooleoyl)phosphatidylcholine (BRPC) has been prepared from dioleoylphosphatidylcholine (DOPC). It is shown that the gel to liquid-crystalline phase transition for BRPC occurs below ca. 5 degrees C and that the motional properties of bilayers of BRPC and DOPC as detected by spin-labeled fatty acids are similar. The ATPase activities of the (Ca2+-Mg2+)-ATPase from rabbit muscle sarcoplasmic reticulum reconstituted with BRPC and DOPC are similar. The brominated lipid quenches the fluorescence of the ATPase and can be used to determine selectivity of lipid binding to the ATPase. We show that there is little selectivity on the basis of fatty acyl chain length. Binding constants for phosphatidylcholines and phosphatidylserines are similar in the absence of calcium, although that for phosphatidylserine decreases in the presence of calcium. Phosphatidylethanolamines binds less strongly than phosphatidylcholines, although the difference is small. The largest difference in binding constants is seen between phosphatidylcholines in the gel and liquid-crystalline phases, with a distribution coefficient of 30 in favor of the liquid-crystalline phase. It is shown that the distribution of the ATPase in mixtures of dipalmitoylphosphatidylcholine and BRPC can be understood in terms of the phase diagram for this mixture of lipids. Activities of the ATPase in the presence of mixtures of lipids can be explained in terms of the relative binding constants obtained from the fluorescence experiments.     

Selective inhibition of human erythrocyte Na+/K+ ATPase by cardiac glycosides and by a mammalian digitalis like factor

Na+/K+ATPase is a transport membrane protein which contains the functional receptor for digitalis compounds. In this work we compare the inhibition curves of Na+/K+ATPase measured by the inhibition of 86Rb uptake in human red blood cells by cardiac glycosides and by an endogenous digitalis like factor (EDLF) extracted from human newborn cord blood. The curves of Na+/K+TPase inhibition show a monophasic shape for ouabain, strophantidin, digitoxin, proscillaridin and EDLF whereas a biphasic shape for ouabagenin, digoxin, digoxigenin and digitoxigenin. All the drugs are potent inhibitors of erythrocyte Na+/K+ATPase with an IC50 ranging from 1.8 x 10(-9) M to 1.4 x 10(-11) M for the higher affinity binding site and from 1.8 x 10(-6) M to 5.5 x 10(-9) M for the lower affinity site. Digitoxigenin is the most active showing the higher active site at 1.4 x 10(-11) M. Ouabain and digoxin have higher affinity compared with their corresponding genins, while digitoxigenin shows a binding site with higher affinity than the respective cardiac glycosides. The increased affinity of the drugs to Na+/K+ATPase may be related to a lipophilic region in correspondence of the carbons 10, 9, 11, 12, 13 of the steroid nucleus, situated in the opposite side with respect of the C-OH-14. The comparison of the inhibition curves and the HPLC profile of newborn EDLF and of the investigated cardenolides suggest that EDLF may be a compound identical or very similar to ouabain.     

Na,K-ATPase and cardiac glycosides: new functions of the known protein

Review of hormonal function of endogenous cardiac steroids. Special attention is paid to recently discovered mechanism of signal transduction from Na,K-ATPase that is due to not a change of ionic gradients but to ouabain-induced alteration of enzyme conformation, that, in turn, results in interaction of the enzyme with intracellular proteins. The data concerning discovery and identification of endogenous cardiac steroids and different isoforms of Na,K-ATPase that have various sensitivity to cardiac steroid, are also considered.     

Interaction of fatty acids with the calcium-magnesium ion dependent adenosinetriphosphatase from sarcoplasmic reticulum

The fluorescence emission spectrum of dansylundecanoic acid is sensitive to the environment and appears at a lower wavelength when the fatty acid is bound to protein than when it is bound to phospholipid. When bound to the (Ca2+-Mg2+)-ATPase of sarcoplasmic reticulum, the emission spectrum can be resolved into separate components assigned to fatty acid bound to protein and to lipid. Efficiency of energy transfer from the tryptophan residues of the ATPase to dansylundecanoic is higher for protein-bound probe than for lipid-bound probe. Fluorescence titrations are consistent with three fatty acid binding sites per ATPase with a Kd of 7 microM, and these sites are postulated to occur at the protein-protein interface in ATPase oligomers. Fatty acid incorporated into the lipid component of the membrane appears to be bound outside the lipid annulus around the protein.     

Characterization,quantitation, similarity evaluation and combination with Na+,K+-ATPase of cardiac glycosides from Streblus asper

Streblus asper Lour. (Moraceae) is a medicinal plant in Asian countries including India and Thailand, possessing activities of anti-tumor, anti-allergy, anti-parasitic and anti-bacterial. In this paper, characterization, quantitation and similarity evaluation of cardiac glycosides in different parts of S. asper were investigated by HPLC-Q-TOF-MS and chemometric methods. Then, the inhibition of Na⁺,K⁺-ATPase activity by the compounds isolated from S. asper was measured. Meanwhile, enzyme kinetics and molecular docking were determined to exhibit the combination modes between cardiac glycosides and Na⁺,K⁺-ATPase. As a result, twenty peaks of cardiac glycosides were assigned. Strophanthidin-3-O-α-l-rhamnopyranosyl-(1 → 4)-6-deoxy-β-d-allopyranoside (1), glucostrebloside (2), strebloside (4) and mansonin (8) with a significant activity of inhibiting Na⁺,K⁺-ATPase (IC₅₀ 7.55–13.60 μM) were chosen for the determination of enzyme kinetics, exhibiting anticompetitive inhibitory characteristics towards Na⁺,K⁺-ATPase. Compound 4 could reasonably bind to the active sites of Na⁺,K⁺-ATPase, proved by molecular docking. Furthermore, the contents of the major compounds in four different parts of S. asper were extremely different, analyzed by chemometric methods, similarity analysis and principle compounds analysis. All these findings indicated that the contents of major compounds in different parts of S. asper were extremely different with a significant activity of inhibiting Na⁺,K⁺-ATPase, providing a reference for determination of effective part and administered dosage. The combination modes between cardiac glycosides and Na⁺,K⁺-ATPase were also revealed by enzyme kinetics and molecular docking, which provided a basis for further study of pharmacological activity.     

Exchange rates and numbers of annular lipids for the calcium and magnesium ion dependent adenosinetriphosphatase

A spin-labeled phospholipid is used to study lipid-protein interactions in the (Ca2+,Mg2+)-ATPase of sarcoplasmic reticulum from muscle. A novel null method is used to decompose composite electron spin resonance spectra into two components, characteristic of immobilized and mobile environments. Calculations based on a random mixing model suggest that protein-protein interactions will be relatively rare in these systems and that the immobilized lipid does not represent lipid trapped between proteins but rather represents annular phospholipid at the lipid-protein interface of the adenosinetriphosphatase. The apparent decrease in the amount of immobilized lipid with increasing temperature is shown to be consistent with lipid exchange between bulk and annulus, characterized by an exchange time of 10(-7) s at 37 degrees C. A minimum number of annular phospholipid sites of 32 and 22 are calculated at 0 and 37 degrees C, respectively.     

Interaction of palytoxin and cardiac glycosides on erythrocyte membrane and (Na+ + K+) ATPase

Palytoxin (PTX), at extremely low concentrations (0.01-1 nM), caused K+ release from rabbit erythrocytes. Among the various chemical compounds tested, cardiac glycosides potently inhibited the PTX-induced K+ release. The order of inhibitory potency (IC50) was cymarin (0.42 microM) greater than convallatoxin (0.9 microM) greater than ouabain (2.3 microM) greater than digitoxin (88 microM) greater than digoxin (90 microM). Their corresponding aglycones, even at 10 microM, did not inhibit the K+ release, but competitively antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex in narrow concentration ranges (IC50 = 0.15-2.4 microM), suggesting that the inhibition of K+ release is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity, and the sugar moiety of cardiac glycosides is involved in the inhibition. On the other hand PTX, at higher concentrations (greater than 0.1 microM), inhibited the (Na+ + K+)-ATPase activity. However, this inhibitory effect of PTX was not antagonized by ouabain. It is suggested that, compared with ouabain, PTX has additional binding site(s) on the (Na+ + K+)-ATPase.     

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.