Confining the Sodium Pump in a Phosphoenzyme Form: The Effect of Lead(II) Ions

Abstract

The effect of Pb²⁺ ions on the Na⁺,K⁺-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Experiments were performed by using the electrochromic styryl dye RH421. It is shown that Pb²⁺ ions can bind reversibly to the protein and do not affect the Na⁺ and K⁺ binding affinities in the E₁ and P-E₂ conformations of the enzyme. The pH titrations indicate that lead(II) favors binding of one H⁺ to the P-E₂ conformation in the absence of K⁺. A model scheme is proposed that accounts for the experimental results obtained for backdoor phosphorylation of the enzyme in the presence of Pb²⁺ ions. Taken together, our results clearly indicate that Pb²⁺ bound to the enzyme stabilizes an E₂-type conformation. In particular, under conditions that promote enzyme phosphorylation, Pb²⁺ ions are able to confine the Na⁺,K⁺-ATPase into a phosphorylated E₂ state.

Introduction

The Na⁺,K⁺-ATPase was the first ion pump to be discovered (1), and it is a fundamental integral membrane enzyme for animal physiology. This enzyme, which belongs to the P-type ATPase family, actively transports sodium and potassium ions against their electrochemical gradients across the plasma membrane in most animal cells by utilizing the free enthalpy of ATP hydrolysis (2–4). The Na⁺ and K⁺ electrochemical gradients are required for basic cellular functions such as maintenance of membrane potential, regulation of cell volume, secondary transport of other solutes, and signal transduction. The molecular mechanism of ion transport in P-type ATPases is usually described by the so-called Albers-Post cycle (5,6). According to this model, the pump can exist in two main conformations: E₁ and E₂. The E₁ conformation has a high affinity for sodium ions, can be phosphorylated by ATP, and presents ion binding sites to the cytoplasm. The E₂ conformation has a high affinity for potassium ions, can be phosphorylated by inorganic phosphate (P_i), and presents ion binding sites to the extracellular aqueous phase. The recently published x-ray crystal structures of the Na⁺,K⁺-ATPase (7,8) support the concept that Na⁺ and K⁺ ions share common binding sites, with the exception of the third sodium ion, which binds to a distinct region.

Lead is a toxic heavy metal that poses a major public health problem around the world. The nervous system, kidneys, and blood are primary targets of the toxic effects of Pb²⁺. At the cellular and molecular level, it is reported that lead has the potential to induce oxidative stress and cause direct damage to the structure and function of biological membranes (9). It is known that several heavy metals, including lead, inhibit the activity of P-type ATPases. For instance, toxic heavy metal ions (Cd²⁺, Hg²⁺, Pb²⁺, Cu²⁺, Zn²⁺) have been shown to affect the activity of Ca²⁺-ATPase (10), Mg²⁺-ATPase, and Na⁺,K⁺-ATPase (11–14). In particular, the inhibitory effect of Pb²⁺ on the Na⁺,K⁺-ATPase has been investigated in different membrane preparations, i.e., microsomes from beef cerebellar cortex (11), human erythrocyte membranes (12), synaptic plasma membranes from rat brain (13), and Electrophorus electroplax microsomes (14).

Despite such experimental evidences, however, the molecular mechanism underlining Na⁺,K⁺-ATPase inhibition by Pb²⁺ is still unclear. Some information about the effect of Pb²⁺ on the phosphorylation and dephosphorylation of the Na⁺,K⁺-ATPase are provided in earlier studies. Siegel and Fogt (14) suggested that Pb²⁺ may act at a single independent binding site to produce or stabilize an enzyme conformation that can be phosphorylated but that cannot catalyze hydrolysis of enzyme phosphate. Swarts et al. (15) have shown that if Pb²⁺ is present during phosphorylation of Na⁺,K⁺-ATPase by ATP, the rate of phosphorylation increases and a phosphointermediate is formed which is insensitive toward K⁺ and ADP. In a previous article, we have investigated the inhibitory effect of Pb²⁺ on the transport cycle of the Na⁺,K⁺-ATPase by combining biochemical and electrical measurements (16). Our results led us to propose that the inhibitory effect of Pb²⁺ on the Na⁺,K⁺-ATPase may be due to an interference with hydrolytic cleavage of the phosphorylated intermediate P-E₂, which occurs in the K⁺-related branch of the pump cycle.

The aim of this work is to unravel the effect of Pb²⁺ on the electrogenic partial reactions of the Na⁺,K⁺-ATPase enzymatic cycle. The experiments were performed by using a fluorescence technique that makes use of the electrochromic styryl dye RH421, whose spectral characteristics are affected by proximity to charge in the membrane (17,18). This method can monitor electrogenic steps within the pump cycle of the protein, and it has been applied in recent studies to identify and analyze the effect of various inhibitors on the reaction kinetics of the Na⁺,K⁺-ATPase (19,20). Our results reveal which conformational states of the enzyme are affected by Pb²⁺ and enable us to propose a molecular mechanism of the Na⁺,K⁺-ATPase inhibition by Pb²⁺.

Materials and Methods

Materials

Magnesium, sodium and potassium chloride, tris(hydroxymethyl)-aminomethane (TRIS), hydrochloric acid, and lead nitrate were obtained from Merck (Whitehouse Station, NJ) at analytical grade. Adenosine-5′-triphosphate disodium salt (ATP, >97%), TRIS phosphate (>99%), ouabain (≥99%), and digitoxigenin (≥99%) were purchased from Fluka (Buchs, Switzerland).

Phosphoenolpyruvate, pyruvate kinase/lactate dehydrogenase suspension, β-nicotinamide adenine dinucleotide (reduced disodium salt hydrate), and L-histidine were purchased from Sigma-Aldrich (St. Louis, MO) at highest quality available.

The electrochromic styryl dye RH421 was obtained from Molecular Probes (Eugene, OR).

Membrane protein preparation

Membrane fragments containing Na⁺,K⁺-ATPase were obtained by extraction from the outer medulla of rabbit kidneys using procedure C of Jørgensen (21). The specific ATPase activity was measured by the pyruvate kinase/lactate dehydrogenase suspension assay (22). Protein concentration was determined by the method of Lowry et al. (23) using bovine serum albumin as a standard. The preparation used in this work had a specific activity of 1700 μmol P_i/(h·mg protein) at 37°C. The total protein content of membrane fragments was 2.07 mg/mL.

Fluorescence measurements

The styryl dye RH421 is an amphiphilic molecule that inserts into the lipid domains of Na⁺,K⁺-ATPase membrane fragments and detects changes of electric field strength inside the membrane dielectric but not conformational transitions (18). Such changes in electric field strength may arise from ion binding or release and translocations in the course of the pump cycle of the protein. The fluorescence level refers to a particular charged state of the enzyme and the fluorescence change between states can be assumed to be proportional to the change of the electric field in the membrane (17,18). The styryl dye responds with a shift of the absorption spectra to longer (red) or shorter (blue) wavelengths corresponding to changes in the local electric potential inside the membrane to more negative or more positive values, respectively (18).

Fluorescence measurements were carried out with a FP-6500 spectrofluorometer (JASCO, Easton, MD). The excitation wavelength was set to 580 nm (slit-width 10 nm) and the emission wavelength was set to 650 nm (slit-width 20 nm). The thermostated cell holder was set at 20°C and was equipped with a magnetic stirrer. A high-pass optical filter (λ_cut = 590 ± 6 nm; Edmund Optics, Barrington, NJ) was put between the cuvette and the detector to cut off contributions to the emitted radiation due to higher harmonics.

If not otherwise stated, the standard buffer solution contained 25 mM histidine (pH 7.2), and 5 mM MgCl_2. After an equilibration time of 10 min, the styryl dye RH421 was added to the buffer, followed by the aqueous suspension of protein containing membrane fragments after further 5 min. The final concentrations were typically 200 nM for the RH421 dye and ∼6 μg/mL for the protein suspension.

Data obtained from each fluorescence experiment were normalized according to the function ΔF/F₀ = (F–F₀)/F₀ with respect to the initial fluorescence level before the first substrate addition, F₀, so that different experiments can be compared easily.

When needed, the experimental data were fitted by the binding isotherm (Hill type function)

$$\Delta F/F_0={\left(\Delta F/F_0\right)}_0+{\left(\Delta F/F_0\right)}_{\max }\left(\frac{c^n}{c^n+K_{0.5}^n}\right)\text{,}$$ (1)

where (ΔF/F₀)₀ is the initial fluorescence level at 0 substrate concentration, (ΔF/F₀)_max is the maximal fluorescence change attained at saturating substrate concentrations, c is the substrate concentration, K_0.5 is the half-saturation concentration, and n is a coefficient related to the cooperativity of the substrate binding process.

For pH titrations, data were fitted by the equation

$$\Delta F/F_0={\left(\Delta F/F_0\right)}_{\mathrm{\infty }}+{\left(\Delta F/F_0\right)}_{\max }\left[\frac{1}{1+{10}^{n\left(\text{pH}-\text{pK}\right)}}\right]\text{,}$$

where (ΔF/F₀)_∞ is the fluorescence level at high pH.

In the presence of ATP, free Pb²⁺ concentration was calculated by the MaxChelator software program (24).

Standard experiment

The typical trace of a standard experiment is reported in Fig. S1 in the Supporting Material (trace a). In standard buffer at pH 7.2 and in the absence of Na⁺ and K⁺, the Na⁺,K⁺-ATPase assumes a proton-bound conformation, H_xE₁ (1.5 < x < 1.8 (25)), that determines a fluorescence level taken as a reference zero. The subsequent addition of 50 mM NaCl shifts quantitatively the pump into the Na₃E₁ conformation. It is now well established that only the third sodium ion binds electrogenically to the pump (26), thus determining the fluorescence decrease (∼20% ΔF/F₀) observed here (Fig. S1, trace a). Sodium occlusion, protein phosphorylation, conformational change from E₁ to E₂, and sodium release on the extracellular side are induced by subsequent addition of 500 μM ATP, which displaces the protein in the P-E₂ conformation. The uncompensated release of three sodium ions is responsible for the observed 60% ΔF/F₀ increase of the fluorescence intensity (Fig. S1, trace a). The final addition of 20 mM K⁺ promotes full turnover conditions, accompanied by a fluorescence decrease (Fig. S1, trace a) due to accumulation of positive charge inside the membrane. The final steady-state fluorescence level is due to the weighted contribution of the ion-bound conformations (20).

Usually, each experiment was repeated at least three times. Traces reported in the figures are representative of the experiment performed.

Backdoor phosphorylation

To perform backdoor phosphorylation, Na⁺,K⁺-ATPase-containing membrane fragments were equilibrated in the standard buffer (25 mM histidine, pH 7.2, and 5 mM MgCl₂) with 200 nM RH421 until a constant fluorescence level was obtained. TRIS phosphate (TRIS-P_i) was then added up to 0.5 mM in appropriate aliquots from a 100-mM aqueous stock solution at pH 7.0 to titrate the pump into its phosphorylated state. The same experiment was repeated after equilibration of the pump with different free Pb²⁺ concentrations. The addition of inorganic phosphate and Pb²⁺ ions did not affect buffer pH.

Titration experiments

After equilibration of the protein in the standard buffer, titrations with Na⁺ in the E₁ conformation were carried out by adding small aliquots of aqueous stock solutions of NaCl (5 M and diluted stocks). Analogous titrations were performed with Na⁺ and K⁺ ions, after confining the pump in the P-E₂ conformation by addition of 0.5 mM TRIS-P_i at pH 7.0 (backdoor phosphorylation). All titrations were carried out in the absence and presence of 10-μM free Pb²⁺.

To perform pH titrations, the protein was equilibrated in a buffer composed by 25 mM His, pH 7.4, and 5 mM MgCl₂ (for titrations in the E₁ conformation) or 25 mM His-TRIS, pH 8.1, and 5 mM MgCl₂ (for titrations in the P-E₂ conformation produced by 50 mM NaCl plus 500 μM ATP). After equilibration, small aliquots of 0.25, 0.5, or 1 M HCl were added to the cuvette, depending on the experiment carried out. The same titration was performed in a second cuvette in which pH was measured by a glass microelectrode (model No. 1093B; Hanna Instruments, Woonsocket, RI). The final pH into the two cuvettes was found to differ by no more than 0.08 pH units.

Results

The effect of Pb²⁺ ions on the Na⁺,K⁺-ATPase was investigated in detail by means of steady-state fluorescence spectroscopy. Previous work has shown that Pb²⁺ inhibits the steady-state hydrolytic activity of the sodium pump in the 0–5 μM range (16). It was proposed that lead ions may interfere with the dephosphorylation step of the enzymatic cycle. The experiments reported in this article were carried out to unravel the mechanism of inhibition at the molecular level by determining the conformational states of the enzyme that are mostly affected by Pb²⁺ ions.

Control experiments

In order to follow protein-related fluorescence emission by the RH421 dye in the presence of Pb²⁺ ions, it is first necessary to check whether lead(II) interferes with the fluorescence emission mechanism of the probe. In particular, Pb²⁺ may interact nonspecifically with the membrane surface, being a divalent ion (27). In this manner, it may produce variations of the local field strength that may be sensed by the electrochromic dye. Therefore, we performed control experiments to exclude possible Pb-induced artifacts.

In our control experiments we used 10 μM free Pb²⁺, which is able to block completely the ATPase activity (K_I = 0.5 μM (16),). We tested the effect of 10 μM free Pb²⁺ on membrane fragments containing a ouabain-inactivated Na⁺,K⁺-ATPase. For this purpose, the protein was incubated in standard buffer in the absence of Na⁺ and K⁺ ions and in the presence of P_i (from TRIS-P_i, pH 7.0), so that the protein is confined in the P-E₂ conformation (28) (Fig. S1, trace b). Under these conditions, ouabain is able to bind to the protein inhibiting the steady-state hydrolytic activity (29). The addition of 100 μM ouabain determines a reduction of the steady-state fluorescence level as compared to that of the P-E₂ level (30). The following addition of 10 μM Pb²⁺ leaves the fluorescence level unchanged (Fig. S1, trace b). An analogous fluorescence pattern was obtained by adding 100-μM digitoxigenin instead of ouabain (not shown). Moreover, the same concentration of lead(II) ions does not influence the fluorescence emission of the styryl dye partitioned in pure lipid vesicles (not shown). Based on these results, we can exclude an interference of Pb²⁺ with the RH421 dye up to 10 μM Pb²⁺.

Effect of Pb²⁺ on ion-bound conformations

We then added 10 μM free Pb²⁺ to the ion-bound conformations of the Na⁺,K⁺-ATPase, i.e., Na₃E₁, E₂(K₂), and H_xE₁ (Fig. 1, traces A–D). It is possible to confine the Na-pump in the Na₃E₁ or E₂(K₂) conformations by adding saturating concentrations of Na⁺ or K⁺ ions to the standard buffer (18,31). On the other hand, at pH 7.2 and in the absence of Na⁺ and K⁺, protons act as a congeneric ion species and produce a H_xE₁ conformation, with 1.5 < x < 1.8 (25).

Figure 1.

Addition of 10 μM Pb²⁺ to the ion-bound conformations Na₃E₁ (trace A), E₂(K₂) (C), and H_xE₁ (traces B and D). (Shading) Preferentially adopted conformation when Pb²⁺ is added. Where indicated (arrows), sodium and potassium ions are added at final concentrations of 50 mM and 20 mM, respectively. The initial fluorescence level, relative to H_xE₁ conformation, is due to RH421 dye partitioned into protein-containing membrane fragments. (Dashed line) Zero fluorescence level taken as reference.

The addition of 50 mM NaCl stabilizes the Na₃E₁ conformation of the pump (18,31), and it is accompanied by a fluorescence decrease that is associated to the electrogenic binding of the third sodium ion (26) (Fig. 1, trace A). On the other hand, the addition of 20 mM KCl produced an E₂(K₂) conformation through the reaction pathway H_xE₁ + 2K⁺ ⇆ K₂E₁ ⇆ E₂(K₂) (18,31). It has been shown that binding of potassium ions on the extracellular side is completely electroneutral at acidic pH, and it exhibits a small electrogenic contribution at higher pH values (25). In particular, at pH 7.2, the E₁ conformation is partially protonated (25) and K⁺ binding determines a partial charge displacement. With our preparation, the addition of 20 mM KCl produced a 10% ΔF/F₀ decrease (Fig. 1, trace C).

The fluorescence levels characteristic of these ion-bound states remained unchanged upon the addition of lead(II) (Fig. 1, traces A and C). This indicates that under these conditions Pb²⁺ ions do not bind to the protein in an electrogenic fashion, and they do not promote release of the bound ions.

On the other hand, it is interesting to observe that when Pb²⁺ ions are added to the membrane fragments suspension before Na⁺ or K⁺ ions, a small (∼4% ΔF/F₀) fluorescence drop is observed (Fig. 1, traces B and D). In both experiments, Pb²⁺ interacts with the H_xE₁ conformation of the pump. The subsequent addition of saturating Na⁺ (Fig. 1, trace B) or K⁺ ions (Fig. 1, trace D) restores the fluorescence levels characteristic for the ion-bound conformations (in Fig. 1, compare trace A with B, and trace C with D). This suggests that the presence of 10 μM free Pb²⁺ do not prevent Na⁺ and K⁺ binding to the pump. Moreover, by titrating the H_xE₁ and the P-E₂ conformations with Na⁺ or K⁺ ions, almost identical K_0.5 and n values were obtained in the absence and presence of 10 μM free Pb²⁺ (Table S1 in the Supporting Material). Therefore, the presence of lead(II) does not affect the affinity of the protein for sodium and potassium ions. It is worth noting that charge measurements also demonstrated that Na⁺ binding to E₁ and release from P-E₂ are not affected by the presence of Pb²⁺ ions up to 20 μM free Pb²⁺ (16).

In summary, these experiments suggest that: 1), Pb²⁺ ions allow binding of Na⁺ and K⁺ to the protein with unaltered affinity; and 2), they can interact with the H_xE₁ conformation of the Na-pump.

Standard experiments

With this in mind, we carried out additions of 10 μM free Pb²⁺ in correspondence to the various fluorescence levels attained during a standard experiment (Fig. 2).

Figure 2.

Representative series of standard experiments showing the traces obtained by adding 10 μM Pb²⁺ in correspondence of the various steady-state fluorescence levels. (Shaded) Preferentially adopted conformation when Pb²⁺ is added. (Arrows) Lead(II) ions are added where indicated. (Dashed line) Zero fluorescence level taken as reference. The standard experiments were carried out under the experimental conditions reported in Materials and Methods.

Pb²⁺ addition to H_xE₁ and Na₃E₁ conformations (Fig. 2, traces A and B) produces the fluorescence changes observed by adding lead(II) to the ion-bound conformations (Fig. 1, traces B and A, respectively). The following part of the standard experiment does not differ from the pattern observed in the absence of Pb²⁺ ions (Fig. S1, trace a). This can be explained by considering the very high stability constant of the Pb-ATP complex (log K(PbATP) = 7.02 (10)). In fact, the addition of 0.5 mM ATP removes all free Pb²⁺ ions from the solution and the standard experiment can proceed as in the absence of Pb²⁺. A similar behavior was obtained by chelating Pb²⁺ with EDTA before addition of Na⁺ (not shown). This result indicates the reversibility of lead(II) binding to the Na-pump.

Pb²⁺ addition to the P-E₂ conformation determines a significant fluorescence drop (∼25% ΔF/F₀, Fig. 2, trace C) that can be ascribed to positive charge accumulation inside the membrane dielectric. In this experiment, a total Pb(NO₃)₂ concentration of 310 μM was added to the standard buffer in order to obtain a 10 μM free Pb²⁺ concentration in the presence of 0.5 mM ATP (see Materials and Methods). The final addition of K⁺ ions determines a further drop of the fluorescence signal. This suggests that K⁺ ions can bind to the pump in the presence of lead(II) ions, in agreement with the results of the K⁺ titrations in the P-E₂ conformation (Table S1).

A concentration of 10 μM free Pb²⁺ was also added in correspondence to the final step of the standard experiment (Fig. 2, trace D). After the addition of K⁺ ions, the pump is in turnover condition, and the final steady-state level is determined by the weighted contribution of the ion-occluded conformations, P-E₁(Na₃) and E₂(K₂) (20) (Fig. S1, trace a). It has been proposed that the Na₃E₁ conformation (32,33) and not the P-E₁(Na₃) (34) contributes to the final fluorescence level. Both sodium-bound conformations are expected to contribute equally to the steady-state fluorescence level, due to the electroneutrality of the phosphorylation process (31). The addition of lead(II) determines an increase of the steady-state fluorescence level (Fig. 2, trace D). It is interesting to note that the final level is the same as that obtained in trace C.

In summary, the standard experiments indicate that:

• 1.Pb²⁺ ions bind reversibly to the Na-pump.
• 2.They also bind to an E₂-type conformation.
• 3.The presence of lead(II) does not prevent K⁺ binding to the protein in the presence of Na⁺ ions and ATP.

pH titrations

We then performed pH titrations of E₁ and P-E₂ conformations in the absence and presence of 10 μM free Pb²⁺. The experiments begin at high pH values and the subsequent HCl additions titrate the unoccupied binding sites, shifting to the right the reaction sequences H_xE₁ ⇆ H₂E₁ ⇆ E₂(H₂) and P-E₂ ⇆ P-E₂(H₂), respectively.

Results for the E₁ conformation are reported in Fig. 3 A. The effect of Pb²⁺ ions is moderate, producing only a slight (5%) reduction of ΔF/F₀ at the initial pH of 7.4. The subsequent HCl addtions titrated the residual empty binding sites. Similar experiments were performed in the P-E₂ state produced by 50 mM NaCl plus 500 μM ATP. In this case, the addition of Pb²⁺ ions causes a major drop of the fluorescence signal at pH 8.1 (Fig. 3 B), similarly to that observed during the standard experiment (Fig. 2, trace C). In particular, it is interesting to observe that in the presence of Pb²⁺ the fluorescence drop over the pH range from 8 to 5 is approximately half that in the absence of Pb²⁺.

Figure 3.

pH titrations performed in the E₁ (A) and P-E₂ (B) conformations, in the absence (solid circles) and presence (open circles) of 10 μM free Pb²⁺. Experimental data were fitted by Eq. 2. (A) pK = 6.8 ± 0.1, n = 0.9 ± 0.2 (no Pb²⁺); pK = 6.56 ± 0.08, n = 1.2 ± 0.3 (with Pb²⁺). (B) pK = 5.57 ± 0.02, n = 1.00 ± 0.04 (no Pb²⁺); pK = 5.87 ± 0.09, n = 1.1 ± 0.2 (with Pb²⁺).

All experimental data can be fitted by the binding isotherm of Eq. 2. In the case of E₁ conformation, the pK values in the absence and presence of Pb²⁺ ions are 6.8 ± 0.1 and 6.56 ± 0.08, respectively. On the other hand, in P-E₂ state, the pK value raises from 5.57 ± 0.02 (no Pb²⁺) to 5.87 ± 0.09 (10 μM free Pb²⁺).

It is worth noting that the fluorescence level at low pH is the same for all titrations independently of the presence of lead(II) ions.

Moreover, by comparing the fluorescence levels of the E₂(H₂) or P-E₂(H₂) (−0.1), H_xE₁ (0.0) and P-E₂ (0.48) states in the absence of Pb²⁺, the number of protons, x, can be estimated to be 1.7 at pH 7.2.

Backdoor phosphorylation

Finally, we investigated the effect of Pb²⁺ on the phosphorylated form of the enzyme. It is known that the Na⁺,K⁺-ATPase can be phosphorylated by inorganic phosphate (P_i) in the presence of Mg²⁺ ions but in the absence of Na⁺ and K⁺ ions (28). The reaction is known as backdoor phosphorylation and it can be split into two reaction steps. In the first step, the enzyme undergoes a conformational transition, H₂E₁ ⇆ E₂(H₂), and in the second step phosphorylation occurs, E₂(H₂) + P_i ⇆ P-E₂H₂ ⇆ P-E₂ (28). In the absence of Na⁺ and K⁺, H⁺ acts as a congeneric ion species because the formation of the occluded state with empty binding sites, E₂(), is energetically unfavorable. The electrogenicity of the backdoor phosphorylation is due to the release of both H⁺ in the P-E₂ conformation, P-E₂H₂ ⇆ P-E₂, because the affinity for protons in the P-E₂ conformation is significantly lower than in the E₁ conformation (25).

Backdoor phosphorylation experiments were carried out as described in Materials and Methods. The experimental data relative to P_i titrations in the presence of 0, 0.1, 1, 2.5, 5, and 10 μM free Pb²⁺ are reported in Fig. 4 (A–F, respectively).

Figure 4.

Backdoor phosphorylation titrations carried out in the presence of different Pb²⁺ concentrations. Experimental data are obtained by averaging two-to-three independent titrations for each concentration of Pb²⁺ ions. (Error bars) Standard deviation (where not visible, they are hidden by the data points). (Solid lines) Simulated curves obtained by using the six-intermediates model of Fig. 6 (upper panel) as described in the text.

Each titration curve was fitted by a Hill type function (Eq. 1), and the characteristic parameters K_0.5 and n were determined (Table 1). In the absence of Pb²⁺ the half-saturation constant for P_i was found to be 12.1 ± 0.8 μM, which is in reasonable agreement with the values of 23 μM (28), 29 μM (35), and 32 μM (36) reported in the literature for the kidney enzyme. The K_0.5 value rises to 21 ± 2 μM when Pb²⁺ is increased to 10 μM (Table 1). This behavior indicates an apparent decrease in affinity of the sodium pump for P_i. As a consequence, a possible competition between P_i and Pb²⁺ binding to the protein cannot be excluded. Furthermore, the Hill coefficient also increases from 0.9 ± 0.1 in the absence of Pb²⁺ to 1.6 ± 0.2 in the presence of 10 μM free Pb²⁺ (Table 1).

Table 1.

Experimental (K_0.5 and n) and simulated (K_0.5) parameters are reported for each concentration of lead(II) ions

Free Pb2+ concentration (μM)	Experimental		Simulated
	K0.5 (μM)	n	K0.5 (μM)
0	12.1 ± 0.8	0.9 ± 0.1	12.1
0.1	15.3 ± 0.7	1.05 ± 0.04	12.5
1	15.4 ± 0.5	1.11 ± 0.04	14.9
2.5	13.8 ± 0.9	1.12 ± 0.07	16.9
5	18 ± 2	1.7 ± 0.2	18.5
10	21 ± 2	1.6 ± 0.2	19.8

Note that a value for the cooperativity coefficient, n, cannot be obtained from the simulation (see text).

The initial fluorescence level of the titration curves is affected by lead(II). This is not unexpected, because a fluorescence decrease caused by Pb²⁺ addition to the H_xE₁ conformation is also evident from experiments carried out on ion-bound conformations (Fig. 1, traces B and D) and from pH titrations in the E₁ conformation in the presence of Pb²⁺ (Fig. 3 A). Such fluorescence decrease must be determined by proton binding to the protein. The dependence of the initial fluorescence levels on Pb-concentration is reported in Fig. 5 A. The experimental data can be fitted by a binding isotherm (Eq. 1) that describes the equilibrium Pb²⁺ + H_xE₁ + yH⁺ ⇆ H_x+yE₁^Pb. The fitting provides a maximal fluorescence drop of ∼4% ΔF/F₀, K_0.5 = 3.1 ± 0.9 μM and n = 2 ± 1 (Fig. 5 A).

Figure 5.

Dependence of the steady-state fluorescence levels of states H_xE₁ (A) and P-E₂ (B) on Pb²⁺ concentration. (Solid lines) Binding isotherms describing the equilibria Pb²⁺ + H_xE₁ + yH⁺ ⇆ H_x+yE₁^Pb (A) and Pb²⁺ + P-E₂ + zH⁺ ⇆ P-E₂^Pb (H_z) (B). Best fitting with Eq. 1 gives the following values: (ΔF/F₀)₀ = 0, (ΔF/F₀)_max = −0.04 ± 0.01, K_0.5 = 3.1 ± 0.9 μM, and n = 2 ± 1 (A); (ΔF/F₀)₀ = 0.187 ± 0.006, (ΔF/F₀)_max = −0.13 ± 0.02, K_0.5 = 2.5 ± 0.4 μM, and n = 2.1 ± 0.7 (B).

The final fluorescence level is also affected by the presence of Pb²⁺ ions as shown in Fig. 5 B. The decrease of the final fluorescence level with increasing Pb²⁺ concentration has to be attributed by H⁺ binding to the phosphorylated enzyme, as suggested by pH titrations in the P-E₂ conformation (Fig. 3 B). The best curve that fits the experimental data is the binding isotherm relative to the equilibrium Pb²⁺ + P-E₂ + zH⁺ ⇆ P-E₂^Pb (H_z). In this case, the maximal variation of ΔF/F₀ is ∼12%. The K_0.5 (2.5 ± 0.4 μM) and n (2.1 ± 0.7) values are almost the same as those determined for Pb²⁺ binding to H_xE₁. The K_0.5 value denotes an affinity for Pb²⁺ very similar to that in the E₁ state. Considering the high uncertainty related to n, it does not make sense to propose a cooperative binding of more than one Pb²⁺ ion to the pump in both equilibria.

Discussion

Fluorescence measurements by means of styryl dyes are useful to characterize electrogenic partial reactions of the Na⁺,K⁺-ATPase (33,37–41) and the pump interaction with drugs (19,20). We recently found that Pb²⁺ ions inhibit the enzyme activity of the sodium pump almost quantitatively at concentration of 10 μM and above (K_I = 0.5 μM (16)). We now use steady-state fluorescence measurements to characterize in detail the effect of Pb²⁺ ions on the Na-pump and to pinpoint the reaction step(s) of the enzymatic cycle at which the heavy-metal ions provoke their inhibitory action.

We first determined the effect of Pb²⁺ on the ion binding process. Our results indicate that Na⁺ and K⁺ ions can bind to the pump also in the presence of Pb²⁺ ions (Fig. 1, traces B and D). Although charge measurements previously indicated that sodium binding and release are not affected by lead(II) ions (16), here we demonstrated for the first time, to our knowledge, that Pb²⁺ ions do not modify the binding affinity of the pump for sodium and potassium ions (Table S1). We can therefore conclude that Pb²⁺ binding to the protein does not block the access pathway to ion-binding sites.

The experiments performed by adding Pb²⁺ to the ion-bound conformations also indicate that lead(II) appears to bind to the H_xE₁ conformation (Fig. 1, traces B and D). Under the adopted experimental conditions (i.e., absence of Na⁺ and K⁺ ions), we observed a slight reduction of the fluorescence signal (∼5%), which is confirmed by pH titrations in the E₁ state (Fig. 3 A) and the experimental data of Fig. 5 A. This reduction has to be ascribed to a corresponding further 5% protonation of the pump, producing an H_x+yE₁^Pb state, with y ≈ 0.1. It is worth mentioning that pH titrations in the E₁ state indicate a Pb²⁺-induced moderate decrease in proton affinity.

On the other hand, a significant fluorescence decrease (∼25%) is observed after addition of Pb²⁺ to the P-E₂ conformation (Fig. 2, trace C). Because Pb²⁺ was added when K⁺ ions were still absent, the species responsible for the decrease in fluorescence can be Na⁺, H⁺, or Pb²⁺ ions themselves. Pb²⁺ ions do not affect the fluorescence level attained by the ouabain-induced P-E₂ state (Fig. S1, trace b), therefore it is unlike that they can be responsible for the fluorescence drop observed in this case. On the other hand, it seems unreasonable to propose a Pb-induced re-binding of Na⁺ ions to the pump, because it is known that the P-E₂ conformation has a low affinity for sodium (2). Therefore, the fluorescence level obtained after the addition of Pb²⁺ to the P-E₂ conformation has to be attributed to a protonated P-E₂ state. We can indicate this conformation as P-E₂^Pb(H_z). From pH titrations of the P-E₂ state (Fig. 3 B), z can be estimated to be ∼1 at pH 7.2. Interestingly, these experiments also reveal that the presence of 10 μM Pb²⁺ increases the pK value and, therefore, the affinity of the pump for H⁺.

Under turnover conditions and in the absence of Pb²⁺, the pump is present partially in an E₁ state with three Na⁺ bound. This causes a lower steady-state fluorescence level (Fig. S1, trace a). In the presence of lead(II), the final level is significantly higher (Fig. 2, trace D), indicating that Pb²⁺-induced sodium-bound states (Na₃E₁^Pb or P-E₁^Pb(Na₃)) are not present. This reveals that the enzyme is almost completely blocked in an E₂-type conformation in which two K⁺ are bound (and no turnover occurs). According to traces A and C in Fig. 2, the fluorescence level of states [H_xE₁ + Pb²⁺] and [P-E₂^Pb(H) + K⁺] are practically the same. This can be explained by two monovalent cations (H⁺ or K⁺) bound to the Pb²⁺-modified pump.

Simulations

To obtain further insight into the mechanism of interaction of Pb²⁺ ions with the Na-pump, we adopted a model scheme of six intermediates for the chemical reactions involved in the backdoor phosphorylation in the presence of Pb²⁺ ions (Fig. 6, upper panel). Given the equilibrium constants for each step of the scheme, K_i, it is possible to solve the resulting linear equations system for the equilibrium concentration of each intermediate at different concentrations of P_i and of free Pb²⁺. The fluorescence data can be reproduced by considering the specific fluorescence level, f_i, of each intermediate (see the Appendix in the Supporting Material).

Figure 6.

(Upper panel) Proposed model scheme for the reactions involved in backdoor phosphorylation in the presence of Pb²⁺. For each step, the relative equilibrium constant, K_i, is indicated. For simplicity, protons are omitted in the scheme. (Lower panel) Distribution of the concentrations for all intermediates against P_i concentration as obtained from the simulation. The concentration of Pb²⁺ is 10 μM. For each curve, the corresponding intermediate is indicated.

In the proposed model scheme, steps 1 and 2 correspond to the backdoor phosphorylation. Steps 3, 4, and 5 refer to binding of Pb²⁺ to the P-E₂, E₂, and E₁ conformations respectively, as discussed above. Steps 6 and 7 are introduced assuming that Pb²⁺ does not prevent the E₁ to E₂ conformational transition nor enzyme phosphorylation.

A satisfying reproduction of the experimental data is obtained by assigning to K_i and to f_i the values reported in Table 2 (Fig. 4, solid lines). The values for the equilibrium constants K₁ and K₂ were taken from Apell et al. (28). In particular, the value of K₂ has been adjusted to obtain the best fit of the experimental data.

Table 2.

Values of K_i and f_i used in the simulation according to the six-intermediates model scheme

i	Ki	Source	Specific fluorescence levels		Source
			fi	Intermediate
1	0.39	Apell et al. (28)	0	E1	By definition
2	8.7 μM	Adapted from Apell et al. (28)	0	E2	By definition
3	5.0 μM	Simulation	+0.19	P-E2	Fig. 5B
4	2.5 μM	Fig. 5A	−0.05	E1Pb	Fig. 5A
5	2.5 μM	Fig. 5A	−0.05	E2Pb	Fig. 5A
6	0.39	See Appendix in the Supporting Material	+0.04	P-E2Pb	Fig. 5B
7	17.4 μM	See Appendix in the Supporting Material

See Fig. 6 for the six-intermediates model scheme.

The equilibrium constants K₄ and K₅ are identical (2.5 μM), to indicate the same affinity of Pb²⁺ ions for the E₁ and the E₂ conformations. This value is in agreement with that determined experimentally, 3.1 ± 0.9 μM (Fig. 5 A). The difference between the two values is negligible, considering the experimental uncertainty.

The value of the K₃ equilibrium is twice that of K₄ and K₅ to obtain the best simulation curves. In fact, by setting K₃ = K₄ = K₅ = 2.5 μM, no variation of the K_0.5 for P_i is observed with increasing Pb²⁺ concentration, whereas a value of K₃ < K₄ = K₅ = 2.5 μM determines an apparent increase of affinity for P_i (not shown), contrary to that observed experimentally.

Finally, the values of K₆ and K₇ are not independent parameters, and are calculated according to the principle of detailed balance (see (42) and Appendix in the Supporting Material).

The specific fluorescence levels of the P-E₂, E₁^Pb, E₂^Pb, and P-E₂^Pb intermediates, that is f₃, f₄, f₅, and f₆, respectively, are related to the degree of protonation of these states and were taken from Fig. 5 (Table 2).

The simulated curves so obtained are in very good agreement with the experimental data (Fig. 4). In particular, the initial and final fluorescence levels nicely agree with the experimental values, as well as the half-saturation constants, K_0.5 (Table 1). Moreover, by assigning a nonzero value to the specific fluorescence levels of the intermediates E₁^Pb and E₂^Pb, we can reproduce the Pb-induced fluorescence drop observed after addition of Pb²⁺ ions to the pump in the absence of any other reagent, i.e., Na⁺, K⁺, P_i, or ATP (Fig. 5 A). However, the reaction scheme of Fig. 6 (upper panel) cannot simulate the increase of the Hill coefficient experimentally observed. This increase of n is probably an apparent effect.

Finally, it can be useful to analyze the concentration distribution for all the intermediates to determine the most stable conformations at equilibrium. As shown in Fig. 6 (lower panel), in the presence of 10 μM Pb²⁺ the preferentially adopted conformation is the P-E₂^Pb form (∼65%) at saturating P_i. On the other hand, the E₂^Pb form is the main species at low P_i.

Mechanism of interaction

In conclusion, with the experiments reported here we analyzed the effects of Pb²⁺ on the electrogenic partial reactions of the Na⁺,K⁺-ATPase enzymatic cycle. Taken together, our fluorescence measurements allow us to propose a possible molecular mechanism for the interaction of Pb²⁺ ions with the Na⁺,K⁺-ATPase.

One important finding of this study is that Pb²⁺ ions do not affect the Na⁺ and K⁺ binding affinities in the E₁ and P-E₂ conformations of the enzyme. Another interesting observation is that Pb²⁺ binds with almost the same affinity to the E₁ and P-E₂ conformations. In the absence of Na⁺ and K⁺ and in the presence of Pb²⁺, an E₁ state is formed with approximately two H⁺ (H_x+yE₁^Pb, x + y = 1.8), whereas in the presence of Na⁺, ATP, and K⁺, Pb²⁺ ions stabilize a P-E₂ state with two K⁺ (P-E₂^Pb(K₂)). It is also worth noting that Pb²⁺ promotes at pH>7 binding of one H⁺ to the P-E₂ conformation in the absence of K⁺ (P-E₂^Pb(H)).

In summary, our experimental data clearly indicate that Pb²⁺ bound to the enzyme stabilizes E₂-type conformations, as also confirmed by the results of the simulation. In particular, under conditions that promote enzyme phosphorylation, Pb²⁺ ions are able to confine the Na⁺,K⁺-ATPase into a phosphorylated E₂ state. This conclusion supports our hypothesis about the possible interference of lead(II) ions with the dephosphorylation step (16). Moreover, it can explain the experimental results previously reported (15), where a Pb²⁺-induced decrease of the dephosphorylation rate constant was observed.

Finally, in addition to the significance of the Pb²⁺ effect with regard to the catalytic mechanism, we consider that stabilization of the (phosphorylated) E₂ conformation of the enzyme by Pb²⁺ ions may be a very useful tool in structural and crystallization studies of cation transport ATPases. In fact, it is well known that, very often, ATPase crystal structures of the highest resolution are obtained by incorporation of one or even two inhibitors, as recently reported in the case of sarcoplasmic reticulum Ca²⁺-ATPase (43).