Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

Schneeberger, Anne; Apell, Hans-Jürgen

doi:10.1007/s002320010051

Cited by 58 publications

(74 citation statements)

References 35 publications

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“…The results of the crystal structure of the SR Ca-ATPase (Toyoshima et al, 2000), which revealed the position of the Ca 2+ binding sites in the middle of the membrane, and of the electrogenicity of all ion-binding steps, Ca 2+ and H + ions (Butscher et al, 1999, Peinelt & Apell, 2002, as well as the similar¯uorescence responses of all nXITC dyes on the investigated partial reactions as shown in this paper, ®t well together. This structural concept of ion-binding sites that are located in the center of the membrane, is in strong contrast to the observations made with the Na,K-ATPase so far: On the cytoplasmic side, binding of only the third Na + ion is electrogenic, i.e., within the protein dielectric; all other ions, be that Na or K , have to bind close to the surface or in wide, water-®lled vestibules that are still part of the dielectric protein surface (Goldshlegger et al, 1987, Bahinski et al, 1988, Rakowski et al, 1990, Domaszewicz & Apell, 1999, Schneeberger & Apell, 2001). In the experiments presented it could be shown unambiguously that various dyes respond di erently on electrogenic Na + binding, which is a strong indication that an ion is being bound within the sensitive length of the styryl chromophores and not in the center of the membrane.…”

Section: Structure-function Relations Deduced From the Experimental Ocontrasting

confidence: 73%

“…If, in contrast, the dye molecules are di using through the lipid phase of the membrane, an increase of the dye concentration would hardly a ect the amplitude of DF/F 0 because the (average) electric ®eld in the membrane is not signi®cantly altered when the ratio of 60 lipids per dye molecule is decreased to 20. On the other hand, it was shown that partial reactions, which contain con-formational transitions but no change of the number of ions inside the protein, did not a ect the¯uores-cence emission, such as E 1 + 2K + ® E 2 (K 2 ) (StuÈ rmer et al, 1991;BuÈ hler & Apell, 1995;Schneeberger & Apell, 2001) or Na 3 E 1 ® P-E 2 Na 3 , which can be observed in high Na + concentrations (StuÈ rmer & Apell, 1992;Heyse et al, 1994). In the case of 2BITC and 2HITC, conformational transitions of the SR Ca-ATPase were also not accompanied by signi®cant uorescence changes (Butscher et al, 1999, Peinelt & Apell, 2002.…”

Section: Detection Mechanism Of the Styryl Dyesmentioning

confidence: 99%

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Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

Pedersen

Roudná

Beutner

et al. 2002

Journal of Membrane Biology

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Abstract. A family of¯uorescent styryl dyes was synthesized to apply them as probes that monitor the ion-translocating activity of the Na,K-ATPase and the SR Ca-ATPase, similar to the widely used dye RH421. All dyes had the same chromophore but they di ered in the length of the spacer between chromophore and polar head, an isothiocyanate group, and in the lengths of the two identical acyl chains, which form the tail of the dye molecules. A number of substrate-dependent partial reactions of both P-type ATPases a ected the¯uorescence intensity, and the magnitude of the¯uorescence changes was used to characterize the usefulness of the dyes for further application. The experimental results indicate that electrochromy is the major mechanism of these dyes. While in the case of the Na,K-ATPase a single dye, 5QITC, showed larger¯uorescence changes than all others, in the case of the SR Ca-ATPase all dyes tested were almost equal in their¯uorescence responses. This prominent di erence is interpreted as a hint that the position of the ion binding sites in both ion pumps may di er signi®cantly despite their otherwise closely related structural features. Quench experiments with spin-labeled lipids in various positions of their fatty acids were used to gain information on the depth of the chromophore of the di erent dyes within the membrane dielectric, however, the spatial resolution was so poor that only qualitative information on the position of the chromophore in the lipid phase could be obtained.

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Section: Structure-function Relations Deduced From the Experimental Ocontrasting

confidence: 73%

Section: Detection Mechanism Of the Styryl Dyesmentioning

confidence: 99%

Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

Pedersen

Roudná

Beutner

et al. 2002

Journal of Membrane Biology

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“…No other ion but Na + was found to bind to the third ion binding site, and the affinity for Na + binding to the third ion binding site is higher than for the second (Schneeberger & Apell, 2001). From these findings it was concluded that the third ion binding site becomes available only after the first two sites are occupied.…”

Section: Discussionmentioning

confidence: 67%

“…9, box III). Binding constants for the Na + ions in absence of MCS factors and at pH 7.0-7.2 were already established before (Schulz & Apell, 1995, Schneeberger & Apell, 2001, and are adopted for this simulation as KNa1 = 1 lM, KNa2 = 0.8 mM, and KNa3 = 0.9 lM. The simulations carried out with this model could not match the experimental data nor even appropriately represent the trends in fluorescence increases as observed in the experiments (not shown), similar to the respective approach described above for the K + and H + pathway.…”

Section: Na + Pathwaymentioning

confidence: 99%

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

2005

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Abstract. 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 E 1 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.

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“…Previously published careful equilibrium titrations have shown in fact that the fluorescence decrease is due to the binding of the third Na + ion to the E 1 conformation, that is, the formation of the E 1 (Na + ) 3 state (39,40). The binding of the first and second Na + ions do not produce any fluorescence change.…”

Section: Discussionmentioning

confidence: 87%

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

2007

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The kinetics of the E 2 f E 1 conformational change of unphosphorylated Na + ,K + -ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E 2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na 2 ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of g50 µM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E 2 :E 2 f E 2 :E 1 f E 1 :E 1 . In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E 2 :E 2 f E 2 :E 1 and E 2 :E 1 f E 1 :E 1 transitions were found to be 10.4 ((1.0) and 0.49 ((0.02) s -1 , respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 ((0.3) s -1 . It was found that ATP acting at a high affinity site (K d ≈ 0.25 µM), stimulated the reverse reaction, E 1 ATP f E 2 ATP, in addition to its known allosteric low affinity (K d ≈ 71 µM) stimulation of the forward reaction, E 2 ATP f E 1 ATP. Na + ,K + -ATPase 1 was the first ion pump to be discovered (1), and it is one of the most fundamentally important enzymes of animal physiology. The electrochemical potential gradient for Na + ions, which the enzyme maintains, is used as the driving force for numerous secondary transport systems. Examples include the Na + channels in the nerve, which allow the action potential to be produced, the Na + -glucose cotransporter, which is responsible for glucose uptake in intestinal cells, and the Na + /Ca 2+ exchanger in the heart, which plays an important role in muscle relaxation. Na + ,K + -ATPase, furthermore, contributes to the osmotic regulation of cell volume and is a major determinant of body temperature. For all of these functions, the enzyme derives its energy from the hydrolysis of ATP, which is coupled to Na + transport.For many years, it has been known that ATP also has an allosteric effect on the enzyme. Under physiological conditions, ATP binds to the E 2 (K + ) 2 conformation of the enzyme and accelerates the release of K + ions to the cytoplasm without undergoing any hydrolysis. This was first demonstrated by Karlish and Yates (2), who measured the rate of the E 2 f E 1 conformational transition, which occurs simultaneously with K + release, via the stopped-flow technique, utilizing the change in the enzyme's tryptophan fluorescence as the means of detection. This stimulation of the E 2 f E 1 transition by ATP has since been confirmed...

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Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

Cited by 58 publications

References 35 publications

Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

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

Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics

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Ion Selectivity of the Cytoplasmic Binding Sites of the Na,K-ATPase: II. Competition of Various Cations

Cited by 58 publications

References 35 publications

Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

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

Allosteric Effect of ATP on Na+,K+-ATPase Conformational Kinetics

Contact Info

Product

Resources

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Allosteric Effect of ATP on Na⁺,K⁺-ATPase Conformational Kinetics