A Comparison Between Calcium and Strontium Transport by the (Ca²⁺ + Mg²⁺)ATPase of the Basolateral Plasma Membrane of Renal Proximal Convoluted Tubules

Abstract

In this work, the utilization of calcium and strontium by the (Ca²⁺ + Mg²⁺)ATPase of the basolateral plasma membrane of renal proximal convoluted tubules were compared. [⁹⁰Sr]Sr²⁺ and [⁴⁵Ca]Ca²⁺ uptake by vesicles derived from this membrane were strictly dependent on ATP and Mg²⁺, and no other nucleotide was able to support the transport. Each cation inhibited the uptake of the other one in a purely competitive fashion (the same Vmax; increased K_0.5), without causing a significant change in the influx rate. These results indicate that both cations bind at the same transport site on the enzyme, facing the cytosolic surface of the cell. The K_0.5 for Sr²⁺ obtained for (Sr²⁺ + Mg²⁺)ATPase activity was 13.1 ± 0.2 µM and for Sr²⁺ uptake was 13.4 ± 0.1 µM. They were higher than K_0.5 for Ca²⁺ obtained for (Ca²⁺ + Mg²⁺)ATPase activity (0.42 ± 0.03 µM) and for Ca²⁺ uptake (0.28 ± 0.02 µM). It is postulated that the lower ATPase affinity for Sr²⁺ is associated with greater steric difficulties for the occupation by this cation of the binding and transport sites, as a consequence of its greater crystal ionic radius (1.13 Å for Sr²⁺ against 0.99 Å for Ca²⁺).

1. Introduction

Calcium transport across the basolateral membranes of proximal tubule cells is mediated, in part, by an ATPase that is stimulated by micromolar Ca²⁺ concentrations in the presence of Mg²⁺ [1,2,3]. The renal (Ca²⁺ + Mg²⁺)ATPase is located in the plasma membrane of basolatera1 membranes of proximal tubule cells, where it plays a central role in the regulation of intracellular Ca²⁺ levels. Proximal tubules are responsible for the reabsorption of most of the ultrafiltered Ca²⁺ in the nephron [4]. Thus, these cells must handle a continuous flux of Ca²⁺ and, at the same time, maintain a very low cytoplasmic Ca²⁺ concentration.

Evidence has been presented [3,5,6] showing that the renal (Ca²⁺ + Mg²⁺)ATPase belongs to the P-ATPase class [7], passing through two principal conformational states (E1 and E2) during its catalytic cycle.

Membrane vesicles from the sarcoplasmic reticulum of skeletal muscle retain a membrane-bound (Ca²⁺ + Mg²⁺)ATPase (SERCA) which is also able to catalyze ATP hydrolysis, stimulated by Sr²⁺ ions, and to transport Sr²⁺ at the expense of ATP hydrolysis [8]. A plasma membrane (Ca²⁺ + Mg²⁺)ATPase (PMCA) with these kinetic characteristics, also able to hydrolyze ATP stimulated by Sr²⁺ and transporting Sr²⁺, has not yet been characterized. Here, for the first time, we are characterizing the Sr²⁺ ion transport and the (Sr²⁺ + Mg²⁺)ATPase activity in the plasma membrane of a cell.

2. Materials and Methods

2.1. Materials

All the reagents of analytical grade were purchased from Sigma Chemical Co. (St. Louis, MO, USA), or Merck (São Paulo, Brazil). The distilled water used to prepare all the solutions was deionized using a Milli-Q system of resins (Millipore Corp., Burlington, MA, USA). Radioactive Pi (³²Pi) was purchased from Instituto de Pesquisas Energéticas e Nucleares (São Paulo, Brazil) as orthophosphoric acid and was purified by extraction in a phosphomolybdate complex with a mixture of 2-butanol/benzene followed by re-extraction into the aqueous phase with ammonium hydroxide and then precipitation as a MgNH₄PO₄ complex. [γ-³²P]ATP (specific activity 10⁴ Bq/nmol ATP) was prepared as previously described by Glynn and Chappell [9]. Ionized Ca²⁺ and Sr²⁺ concentrations were calculated using a computer program based on an iterative method that takes into account the different species involved in the equilibrium between EGTA, Ca²⁺, Sr²⁺, ATP⁴⁻, Mg²⁺, H⁺, and Pi [10,11].

2.2. Preparation of Purified Vesicles Derived from Basolateral Membranes

Basolateral membrane vesicles were isolated from sheep kidney proximal tubules by the Percoll gradient method [12]. Compared with the initial homogenate, this plasma membrane fraction was enriched 9–12-fold in the specific activity of the basolateral membrane marker (Na⁺ + K⁺)ATPase. Protein concentrations were determined using Folin’s phenol reagent [13] and bovine serum albumin as a standard.

2.3. ⁴⁵Ca²⁺ and ⁹⁰Sr²⁺ Uptake

Except when otherwise noted, the basic medium contained, in a final volume of 0.5 mL, 30 mM Tris-HCl buffer (pH 8.5), 5 mM ATP, 5 mM MgCl₂, 1 mM ouabain, 10 mM NaN₃, 0.1 mM EGTA, and 0.1 mM ⁴⁵CaCl₂ (10 µM free Ca²⁺; specific activity 5.0 × 10³ Bq/nmol CaCl₂) or 0.1 mM ⁹⁰SrCl₂ (10 µM free Sr²⁺; specific activity 5.0 × 10³ Bq/nmol SrCl₂). The experiments were carried out at 37 °C. ⁴⁵Ca²⁺ or ⁹⁰Sr²⁺ uptake was started by the addition of membranes (protein concentration 0.2 mg/mL) and stopped by Millipore filtration [14], using 0.45 µm pore size filters. The ⁴⁵Ca²⁺ and ⁹⁰Sr²⁺ remaining in the vesicles were counted in a liquid scintillation counter after the filters were washed twice with 10 mL of a cold solution containing 2 mM La(NO₃)₃, 100 mM KC1, and 20 mM MOPS-Tris (pH 7.0). ⁹⁰SrCl₂ and ⁴⁵CaCl₂ were obtained from New England Nuclear Corporation (Boston, MA, USA).

2.4. (Ca²⁺ + Mg²⁺)ATPase and (Sr²⁺ + Mg²⁺)ATPase Activities

The activities were measured under the same conditions as the ⁴⁵Ca²⁺ and ⁹⁰Sr²⁺ uptakes, except that the reactions were performed in the presence of 5 mM [γ-³²P]ATP. The reaction was initiated by the addition of membrane and stopped by the addition of 1.0 mL of ice-cold 25% charcoal in 0.1 M HCl to adsorb the non-hydrolyzed [γ-³²P]ATP [15]. Following centrifugation at 4000× g for 30 min, an aliquot of the supernatant was withdrawn to measure the amount of ³²Pi released. Spontaneous hydrolysis of [γ-³²P]ATP was measured in tubes run in parallel in which the enzyme was added after the ice-cold charcoal suspension. The (Sr²⁺ + Mg²⁺)ATPase and (Ca²⁺ + Mg²⁺)ATPase activities were quantified as the difference between the ATP hydrolysis measured in the presence of SrCl₂ or CaCl₂, and in its absence (EGTA 1 mM). ⁴⁵Ca, ⁹⁰Sr, and ³²Pi were counted in a liquid scintillation counter.

2.5. Data Analysis

Data analysis was performed using the software Enzfitter (Elsevier-Biosoft, Cambridge, United Kingdom). Values for the variables were calculated by non-linear regression. Data points represent means ± SEM of three determinations using different membrane preparations. When indicated, the data were analyzed using Student’s t-test, using Prism computer software (GraphPad Software Inc., San Diego, CA, USA). The statistically significant difference was set at p < 0.05.

3. Results and Discussion

3.1. Ca²⁺ and Sr²⁺Transport

In order to determine whether Ca²⁺ and Sr²⁺ are actively accumulated in these plasma membrane vesicles, the samples were incubated with ⁹⁰SrCl₂ or ⁴⁵CaCl₂ in the presence and absence of ATP, in the presence of a non-hydrolyzable analog of ATP, (5′-adenylyl methylene diphosphonate, AMP-P-C-P), and in the presence and absence of MgCl₂. Figure 1 shows the time course of ⁹⁰Sr²⁺ (A) and ⁴⁵Ca²⁺ (B) uptake. The transport rates of ⁹⁰Sr²⁺ and ⁴⁵Ca²⁺ by the basolateral membrane vesicles were very slow when compared to those of the sarcoplasmic reticulum [8]. The maximum accumulation level was reached after four hours, which probably reflects the small number of transport units present in the plasma membrane in most cells. At all times, Sr²⁺ uptake was 30–35% lower than Ca²⁺ uptake, and the difference was probably due to a slower rate of active Sr²⁺ influx as a consequence of steric difficulties for the occupancy of the binding and transport sites, because of its greater crystal ionic radius (1.13 Å for Sr²⁺ against 0.99 Å for Ca²⁺) [16]. In these experiments, in the absence of ATP, about 2.1 nmol ⁹⁰Sr²⁺ × mg protein⁻¹ and 3.6 nmol ⁴⁵Ca²⁺ × mg protein⁻¹ were observed. The same low amounts of accumulated ⁴⁵Ca or ⁹⁰Sr were obtained in the presence of AMP-P-C-P or in the absence of MgCl₂. It can also be seen in this figure that the addition of A23187, a Ca²⁺ ionophore, induces a rapid and total release of ⁹⁰Sr²⁺ and ⁴⁵Ca²⁺ accumulated by the vesicles, which indicates that ⁹⁰Sr²⁺ as well as ⁴⁵Ca²⁺ are found in the luminal compartment.

One of the characteristics of plasma membrane (Ca²⁺ + Mg²⁺)ATPases is the high specificity for the energy-donor substrate used for transport, in this case, ATP [17,18]. As shown in

Table 1. Specificity for different energy-donor substrates in ⁹⁰Sr²⁺ and ⁴⁵Ca²⁺ uptake assays.

Substrates	Uptake of 90Sr (%)	Uptake of 45Ca (%)
ATP	100	100
CTP	2.4	5.2
GTP	2.7	1.4
ITP	1.6	4.5
UTP	1.4	2.7

Abbreviations: ATP: Adenosine triphosphate; CTP: Cytidine triphosphate; GTP: Guanosine triphosphate; ITP: Inosine triphosphate; UTP: Uridine triphosphate.

, both ⁴⁵Ca²⁺ transport and ⁹⁰Sr²⁺ transport present the same pattern of selectivity for the different substrates tested.

The dependence of Sr²⁺ and Ca²⁺ uptake on ATP concentration (Figure 2, panel A and panel B, respectively) presents the same stimulatory profile for both ⁹⁰Sr²⁺and ⁴⁵Ca²⁺ uptake with a Km for ATP in the Sr²⁺ transport curve of 0.65 ± 0.06 mM and a Km for ATP in the Ca²⁺ transport curve of 0.52 ± 0.06 mM, without significant differences between these values (p > 0.05; Student´s t-test).

3.2. Dependence of Sr²⁺ and Ca²⁺ Uptake on Orthophosphate Concentration

As seen in Figure 3, panels A and B, the dependence on Pi concentration presents the same stimulatory profile for both ⁹⁰Sr²⁺ and ⁴⁵Ca²⁺ uptake. Due to the small volume of the basolateral membrane inside-out vesicles, the accumulated Ca²⁺ and Sr²⁺ can reach sufficiently high concentrations in the lumen of the vesicles to saturate the low-affinity site facing the internal surface of the vesicle, leading to inhibition of the hydrolytic activity of the (Ca²⁺ + Mg²⁺)ATPase [3]. There are several observations showing that the addition of anions such as phosphate (Pi) and oxalate increases the amount of Ca²⁺ accumulated inside plasma membrane and inside sarcoplasmic reticulum membrane vesicles [19]. This increase in the amount of accumulated Ca²⁺ is attributed to the fact that the intravesicular anions form insoluble complexes with Ca, thus reducing the concentration of free Ca²⁺ in the lumen and thus decreasing the occupancy of the low-affinity inhibitory site. As seen in Figure 3, panels A and B, the dependence on Pi concentration presents the same stimulatory profile for both Sr²⁺ and Ca²⁺ uptake with the same K_0.5 (5.3 mM and 5.1 mM, respectively; Student’s t-test), reaching saturation at a concentration of 40 mM.

Another possibility that has been suggested is that incubation of the vesicles in media containing potassium phosphate in the pH range 7.0–7.4 would lead to an asymmetric distribution of the anionic species of the buffer due to differences in permeability of H₂PO₄⁻ and HPO₄²⁻ with the generation of a ΔpH with increased Ca²⁺ uptake [3], due to an increase in the intravesicular concentration of H⁺ available for exchange for external Ca²⁺. The existence of Ca²⁺/nH⁺ cotransport was shown in the (Ca²⁺ + Mg²⁺)ATPase of red blood cells [17].

3.3. Dependence of Sr²⁺ and Ca²⁺ Uptake on Free Sr²⁺ and Ca²⁺ Concentrations, Respectively

The mechanism of transepithelial Ca²⁺ transport by renal tubular plasma membrane (Ca²⁺ + Mg²⁺)ATPases presupposes that the system located in the membrane is stimulated by micromolar concentrations of the ion, since cytosolic Ca²⁺ concentrations vary in this range. The dependence of Sr²⁺ uptake on Sr²⁺ concentrations follows Michaelis–Menten kinetics, reaching a maximum velocity of 0.11 nmol ± 0.02 nmol Sr²⁺ × mg⁻¹ × min⁻¹ with a K_0.5 for Sr²⁺ of 13.4 ± 2.4 µM (Figure 4A). As can be seen in Figure 4B, the dependence of Ca²⁺ uptake on Ca²⁺ concentrations follows the same kinetics, reaching a maximum velocity of 0.15 nmol ± 0.04 nmol Ca²⁺ × mg⁻¹ × min⁻¹ with a K_0.5 for Ca²⁺ of 0.28 ± 0.03 µM (Figure 4B). These data show that although the Ca²⁺ active transporter system present in the basolateral plasma membrane can also transport Sr²⁺ with the same velocity, its affinity is greater for Ca²⁺, a cation that has a smaller crystalline ionic radius than Sr²⁺ [16], and which would therefore be able to more easily accommodate the binding located in transmembrane domains of the ATPase molecule [20].

3.4. Sr²⁺ and Ca²⁺ Transport Competition Assays

If the Ca²⁺ transport system present in the basolateral membrane were the same as that responsible for Sr²⁺ transport, the addition of increasing concentrations of non-radioactive Ca²⁺ or Sr²⁺ should decrease the amount of transported ⁹⁰Sr²⁺ or ⁴⁵Ca²⁺, respectively. In Figure 5A, it is observed that ⁹⁰Sr²⁺ uptake decreases as increasing concentrations of free Ca²⁺ are added simultaneously. The same inhibitory effect can also be observed on ⁴⁵Ca²⁺ uptake when increasing concentrations of free Sr²⁺ are added to the reaction medium (Figure 5B). However, the inhibition promoted by the addition of Sr²⁺ only becomes more evident when a concentration of Sr²⁺ 100 times greater than that of Ca²⁺ is added to the medium. These results are consistent with those showing that the transporter system present in the plasma membrane has greater affinity for Ca²⁺ than for Sr²⁺.

3.5. Inhibition of Sr²⁺ Transport by Vanadate

It has been demonstrated that orthovanadate is capable of inhibiting a large number of transporting ATPases that form a phosphorylated intermediate during their catalytic cycle, defined as P-ATPases [7]. Vanadate is one of the most studied P-type ATPase inhibitors [21]. It is known that the orthovanadate ion VO₃^- presents a great structural similarity to PO₄^2-. Figure 6 shows the effects of vanadate on (Sr²⁺ + Mg²⁺)ATPase and (Ca²⁺ + Mg²⁺)ATPase activities of the renal basolateral membrane. Using plasma membranes, the (Sr²⁺ + Mg²⁺)ATPase and (Ca²⁺ + Mg²⁺)ATPase activities are similarly very sensitive to vanadate below 10 μM of the compound (Figure 6, panels A and B). The same inhibitory profile is observed when the concentration of vanadate in Sr²⁺ and Ca²⁺ uptake assays is increased (Figure 7, panels A and B).

3.6. pH Dependence Assays

Several amino acids in different domains of the (Ca²⁺ + Mg²⁺)ATPase molecule are potential candidates to offer -COOH and -OH groups in their side chains which are capable of contributing to the binding of the Ca²⁺ or Sr²⁺ ions after deprotonation [22]. With the use of renal plasma membrane vesicles, low levels of Sr²⁺ and Ca²⁺ accumulation (Figure 8, panels A and B) were observed at pH 5.0. However, a progressive increase in the velocity of accumulation of both cations was observed due to the increase in pH of the medium. In Figure 9, it is observed that (Sr²⁺ + Mg²⁺)ATPase (Figure 9A) and (Ca²⁺ + Mg²⁺)ATPase (Figure 9B) activities increase as the pH of the medium increases, with a profile similar to that shown for Sr²⁺ and Ca²⁺ uptake in Figure 8, panels A and B.

3.7. Specificity for Different Energy-Donor Substrates

It has been described that one of the differences between plasma membrane (Ca²⁺ + Mg²⁺)ATPase (PMCA) and sarcoplasmic reticulum (Ca²⁺ + Mg²⁺)ATPase (SERCA) is the high specificity for ATP [6]. Nucleotides other than ATP are inefficient to sustain Ca²⁺ uptake catalyzed by different plasma membrane (Ca²⁺ + Mg²⁺)ATPases. The high specificity for ATP as an energy donor for Sr²⁺ and Ca²⁺ transport (Table 1), confirms the plasma membrane origin of the vesicles used in this study, and rules out the possibility that Sr²⁺ uptake was due to endoplasmic reticulum membrane vesicles present in the preparation used.

The reaction medium contained 10 μM free Ca²⁺ or 10 μM free Sr²⁺ and 5 mM of the nucleotides indicated in the Table 1. The reaction mixtures were incubated for 30 min at 37 °C.

3.8. Conclusions

From the results described and discussed in Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8 and Figure 9, we propose (Figure 10) that the electronic configuration of Sr²⁺ perturbs its proper alignment with the aspartic acid (D895), asparagine (N891), glutamic acid (E433), and methionine (M894) residues of the (Ca²⁺ + Mg²⁺)ATPase plasma membrane molecule and, consequently, the kinetic properties of its transit through the channel formed by the transmembrane TM4, TM5, and TM6 domains [20]. These results can help explain how the electronic configuration underlies modifications of cation flux properties across biological membranes. Mutation of the residues above to investigate whether Sr²⁺ transport still occurs constitutes a future direction for this study.