Probing the extracellular release site of the plasma membrane calcium pump

Wanyan Xu, Betty Jo Wilson, Lin Huang, Emma L. Parkinson, Brent J. F. Hill, and Mark A. Milanick

Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65212


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The plasma membrane Ca2+ pump is known to mediate Ca2+/H+ exchange. Extracellular protons activated 45Ca2+ efflux from human red blood cells with a half-maximal inhibition constant of 2 nM when the intracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreased the IC50 for extracellular Ca2+ from ~33 to ~6 mM. Changing the membrane potential by >54 mV had no effect on the IC50 for extracellular Ca2+. This argues against Ca2+ release through a high-field access channel. Extracellular Ni2+ inhibited Ca2+ efflux with an IC50 of 11 mM. Extracellular Cd2+ inhibited with an IC50 of 1.5 mM, >10 times better than Ca2+. The Cd2+ IC50 also decreased when the pH was raised from 7.1 to 8.2, consistent with Ca2+, Cd2+, and H+ competing for the same site. The higher affinity for inhibition by Ni2+ and Cd2+ is consistent with a histidine or cysteine as part of the release site. The cysteine reagent 2-(trimethylammonium)ethyl methanethiosulfonate did not inhibit Ca2+ efflux. Our results are consistent with the notion that the release site contains a histidine.

calcium ion; red blood cell; calcium ion adenosinetriphosphatase; membrane potential


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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THE PLASMA MEMBRANE Ca2+ pump (PMCA) is one of the three primary mechanisms for the removal of cytoplasmic Ca2+ and thus often plays a pivotal role in terminating signal transduction in many cells (1). The PMCA is a member of the P-type pump family. All of these proteins couple the hydrolysis of ATP to the uphill movement of ions; during a portion of each catalytic cycle, the terminal phosphate of ATP is covalently bound to the pump (18). These proteins have similar putative structures; similar kinetic models describe most of the functional data (18). The PMCA offers two advantages for structure-function studies of P-type pumps. The PMCA is composed of a single subunit, in contrast to the Na+-K+ pump, which requires at least two subunits. Portions of PMCA are accessible from the extracellular media, in contrast to the sarco(endo)plasmic reticulum Ca2+-ATPase pump, which is not.

The Ca2+ pump not only mediates Ca2+ efflux, but also proton (H+) influx. The H+ influx in red blood cells was initially inferred from indirect experiments (6, 21, 25, 26, 31). We have used a pH stat technique to measure directly Ca2+/H+ exchange in intact red blood cells (19). We estimated the stoichiometry as 1 Ca2+-2 H+ at extracellular pH 6.2. Recently, Hao et al. (11) have used fluorescent dyes to measure Ca2+, H+, and membrane potential (Em) changes in a reconstituted proteoliposomal system containing purified red blood cell Ca2+ pumps. They observed changes of pH and Em during Ca2+ pump activity at both 30 and 12°C. The stoichiometry, 1 Ca2+-1 H+, was best estimated at 12°C. The Ca2+ pump-mediated Em changes were dependent on the detergent used in the reconstitution. The differences in the conditions may account for the following different observations: 1) the temperatures were different (37 vs. 12°C), 2) the extracellular pH values were different (6.2 vs. 7.2), and 3) the lipid milieu was different (native red blood cells vs. reconstituted proteoliposomes). It is possible that H+ movement is not rigidly coupled and rate-limiting for enzyme turnover but rather reflects the protonation state of key residues in the Ca2+ binding pocket. The pK values of these residues would be expected to change with Ca2+ binding, with conformational changes of the pump, and with temperature. Ca2+/H+ exchange mediated by the Ca2+ pump has also been reported in several other cell types (2, 8, 28).

Recent exciting developments suggest that the Na+ pump has an extracellular high-field access channel. The Na+ pump is in the same family as the PMCA and, in contrast to the Ca2+ pump, the potential dependence has been studied extensively (3, 9, 12, 23, 27, 29). In one version of the high-field access channel model, the first Na+ to exit the pump moves through a high-field access channel (23, 29). Most of the potential drop across the protein (the transmembrane potential) occurs across this high-field access channel in this conformation. Thus the movement of the charged Na+ is influenced by Em. After the first Na+ has come out, the protein changes conformation such that there is a low-field access channel. The exit of the last two Na+ is relatively potential independent because the ions are moving through a low-field access channel.

The fine detail of this model has required impressive technical developments that have allowed very fast time resolution transient charge movement studies (12, 32). Such measurements are not yet technically possible for the PMCA. However, the early development of the access channel model relied upon innovative steady-state measurements; clearly, without those measurements, there would be substantial ambiguity in interpreting the transient measurements. In this report, we use steady-state 45Ca2+ flux measurements to determine whether the PMCA behaves as if it also has an access channel, as might be expected from the similar overall structure of the two proteins.

Studies of H+ interactions with the PMCA are greatly facilitated by use of a pH clamp so that extracellular H+ is varied at fixed intracellular H+ and fixed Em (5, 20). This is possible in red blood cells by treating the cells with DIDS. DIDS inhibits Cl-/HCO-3 exchange (as well as one-half of the Cl- conductance; see Ref. 14). The residual H+ flux is very small, the red blood cell volume is modest, and the intracellular buffer capacity of Hb is substantial. Thus the intracellular and extracellular pH values can be clamped independently and maintained during the flux measurement (20, 35).

In this paper, we characterize several important features of the extracellular site of the Ca2+/H+ exchange pump. We provide the first measurement of the half-maximal inhibition constant (K1/2) for extracellular H+ activation, quantitatively examine the competition between extracellular H+ and divalent cations, and test the access well model (33, 34). In addition, our data suggest indirectly that a histidine may be involved in the extracellular release site.


    METHODS
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Ca2+ efflux from intact red blood cells. Blood was obtained from a consenting volunteer in a heparinized tube. After removal of plasma and white blood cells, the red blood cells were washed three times in a solution of 165 mM NaCl. Cells were stored for up to 5 days. On the day of the measurement, the cells were incubated in a solution containing 150 mM NaCl, 20 mM HEPES, and 10 mM glucose for 30 min and were treated with 100 µM DIDS in the same media for 15 min. The cells were then loaded with 45Ca2+ as previously described (19). Briefly, the cells were incubated at 20% hematocrit and stirred on ice in a solution containing 150 mM NaCl, 20 mM HEPES, 10 µM A-23187, and 45CaCl2 for 30 min. In Figs. 1 and 2B, the loading solution contained 0.1 mM Ca2+ and 0.05 mM Mg2+; in Fig. 2A, the loading solution contained 0.5 mM Ca2+ and 0.25 mM Mg2+; in Figs. 3-9, the loading solution contained 1 mM Ca2+ and 0.5 mM Mg. Next, the cells were washed four times in a solution of 150 mM NaCl, 20 mM HEPES, and 0.2% BSA and then three times in 150 mM NaCl and 20 mM HEPES. The rate of 45Ca2+ efflux was determined by injecting packed cells using a viscous pipetter (Rainin, Woburn, MA) in a stirred thermostated chamber at 37°C. Four samples were taken <1.5 min for the 0.1 mM loaded cells, <2.5 min for the 0.5 mM loaded cells, and <5 min for the noninhibited 1 mM loaded cells. We have found some variability in the ability to remove all of the ionophore, as evidenced by either a very fast efflux or high time 0 intercepts. To determine that the ionophore was removed, we usually included a control efflux in either 110 mM CaCl2 or 5 mM 2-aminoethyl methanethiosulfonate (MTSEA). If 110 mM CaCl2 (at pH 7.2) inhibited <1/2 of the Ca2+ efflux, the experiment was discarded. [The IC50 for extracellular Ca2+ at pH 7.2 is ~30-40 mM (see Table 1 and RESULTS); 110 mM will stimulate 45Ca2+ efflux if ionophore is present.] In latter experiments, we found that MTSEA was an even better control. In all of the experiments in which MTSEA controls were done, the experiment was rejected if the flux with MTSEA was >0.2 of the control flux (at pH 7.2). The residual flux in the presence of MTSEA probably reflects a Ca2+ leak pathway; the highest residual fluxes were observed in cells loaded with the highest Ca2+.


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Fig. 1.   Extracellular H+ activation of Ca2+ efflux from DIDS-treated cells. Results of 2 separate experiments are shown. Cells were treated with DIDS so that the intracellular pH was clamped, and only the extracellular pH varied. Data were fit to the Michealis-Menton equation with half-maximal inhibition constant (K1/2) = 1.74 ± 0.31 nM.




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Fig. 2.   Extracellular Ca2+ inhibition is altered by extracellular H+ and not by membrane potential (Em). Cells were treated with DIDS so that the intracellular pH was clamped; therefore, only the extracellular pH varied. A: Em was varied by replacing Na+ (triangle ) with K+ (open circle ) at an extracellular pH of 7.9 in the presence of 2 µM valinomycin. The average of 3 separate, paired experiments is shown (the SD are within the symbols unless indicated). In these same 3 experiments, the Em was estimated by using [3H]triphenylmethylphosphonium (TPP). In the 3 cases, Em changed 76, 55, and 54 mV. The IC50 values were 3.63 ± 0.17 and 4.16 ± 0.16 mM. B: In a single paired experiment, the IC50 for Ca2+ was lower at an extracellular pH of 8.2 (IC50 = 5.9 ± 1.1) than at an extracellular pH of 7.2 (IC50 = 33.1 ± 3.3), consistent with a K1/2 for H+ of 2 nM.

Em experiments. The Ca2+ efflux was measured in media containing either 132 mM NaCl, 40 mM N-[tris(hydroxymethyl)methyl]3-aminopropanesulfonate (TAPS), pH 7.9, and 2 µM valinomycin or 132 mM KCl, 40 mM TAPS, pH 7.9, and 2 µM valinomycin. The Em was determined on cells loaded in parallel as those containing 45Ca2+, except for the addition of 45Ca2+. The cells were incubated in four different concentrations of triphenylmethylphosphonium (TPP), and the uptake of [3H]TPP was determined following the technique of Freedman and Novak (7). The distribution of [3H]TPP was determined at 2-2.5 min, the same time as the last samples for the flux measurements. In other experiments (and in agreement with Ref. 7), we found that the TPP distribution was within 80% of its final value at this time point. The change of Em was estimated by determining the ratio of the extracellular [3H]TPP in the Na+ medium and the K+ medium at the same total intracellular TPP content. This approach avoids any need to measure or estimate the free intracellular TPP (7).

MTSEA permeability studies. One hundred microliters of packed red blood cells were incubated in 2 ml of a solution containing 150 mM NaCl and 20 mM HEPES in the presence or absence of 2.5 mM MTSEA or 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET) for 10 min at room temperature. The cells were washed three times with 165 mM NaCl. One milliliter of 10% perchloric acid was added to the cell pellet. After resuspension, the solution was centrifuged to pellet the Hb, etc. The supernatant was removed and neutralized by the addition of 1 ml of 200 mM HEPES (pH 7.4) and 1 ml of 1 M 2-amino-2-hydroxymethyl-1,3-propanediol; 1 ml of 5,5'-dithio-bis(2-nitrobenzoate) (DTNB) was added, and the absorbance at 412 nm was determined. We found that MTSEA significantly reduced the amount of DTNB reactive material (presumably glutathione) but that MTSET had little or no effect.

Calculations. H+ activation data were fit to the Michealis-Menton equation [flux = maximal velocity of H+/(K1/2 + H+)] using Kaliedagraph Software (Synergy Software, Reading, PA). IC50 data were fit to the equation fractional flux = IC50/(IC50 + I) where I was the concentration of inhibitor using Kaliedagraph Software. Cd2+ will complex with Cl-; our data are reported as the total Cd2+ in solution; the free Cd2+ will be ~<FR><NU>1</NU><DE>5</DE></FR> of the total (30). Cd2+ may also bind to OH-; thus, the free concentration is a smaller fraction of the total at pH 8.1 than at pH 7.2, and the change in IC50 for total Cd2+ would underestimate the effect of H+/OH- on the IC50 for free Cd2+. In Figs. 2A, 3, and 7 showing the average and SE of n = 3 experiments, we used all of the normalized data from all three experiments for the fit but plotted the average value at each concentration for convenience.

Materials. All reagents were obtained from Fisher Scientific (Springfield, NJ), Sigma Chemical (St. Louis, MO), or Toronto Biochemicals (Toronto, Canada).


    RESULTS
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INTRODUCTION
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Extracellular H+ activation and Em. Extracellular H+ is a substrate for a Ca2+/H+ exchange system. In red blood cells, we are able to vary the extracellular pH at fixed intracellular pH (pH clamp) by treating the red blood cells with DIDS. Extracellular H+ was varied from 0.06 to 70 nM (pH 10.2-7.1; Fig. 1). The K1/2 for extracellular H+ was very low (~2 nM). This H+ activation probably reflects H+ binding to the transport site and thus provides an important test for probes of the site.

The PM Ca2+-H+ pump is in the same family as the Na+-K+ pump. Extensive studies on the voltage dependence of the Na+-K+ pump are consistent with an access well for extracellular Na+ release. An access well model predicts that the IC50 for extracellular Ca2+ inhibition will be Em dependent.

The Em was varied by replacing extracellular Na+ with K+. In Ca2+-loaded red blood cells, the Ca2+-activated K+ channel is active. Valinomycin (2 µM final, hematocrit = 9%) was added as further insurance that Em approx  EK (EK is the Nernst potential for K+). In these experiments, the change of Em was verified by measuring the distribution of [3H]TPP on the same day as the IC50 was determined. The ratio of the outside TPP in Na+ and in K+, at constant inside total TPP, provides an estimate of the change of Em, independent of any intracellular binding of TPP (7). In the three experiments shown, the Em changed by 76, 55, and 54 mV. In these experiments, MTSEA inhibited the control Ca2+ efflux by >90%, indicating that the ionophore was completely removed. As shown in Fig. 2A, changes of Em had little effect on the IC50 for extracellular Ca2+ at pH 7.9 in contrast to the results predicted by the simple access well model.

Interaction of extracellular H+ and Ca2+. Figure 2B also confirms previous data that extracellular Ca2+ is a weak inhibitor of Ca2+ efflux at pH 7.2 (16, 35). Ca2+/H+ exchange models predict that changes of H+ will increase the ability of extracellular Ca2+ to inhibit. At an extracellular pH of 8.2 (6 nM), the IC50 decreased substantially, consistent with Ca2+ competing with a H+ site with a K1/2 of ~2 nM.

The model of extracellular H+ and Ca2+ competing for a common transport site also predicts that extracellular Ca2+ will alter the K1/2 for H+. We chose to determine the K1/2 for H+ under approximately physiological concentrations of Ca2+ and Mg2+ (1 mM each). We found that 1 mM Ca2+ and Mg2+ inhibited Ca2+ efflux at H+ values <10 nM, as expected for Ca2+ and H+ competition at high pH (Fig. 3). The H+ activation in the presence and the absence of 1 mM Ca2+ and 1 mM Mg2+ was adequately fit by a simple hyperbolic curve, i.e., consistent with a Hill coefficient of 1. These data also show that H+ competes with Ca2+ as expected if both H+ and Ca2+ bind to the extracellular transport site.


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Fig. 3.   K1/2 for extracellular H+ activation is increased in the presence of 1 mM Ca2+ and 1 mM Mg2+. Cells were treated with DIDS so that the intracellular pH was clamped; therefore, only the extracellular pH varied. Data were fit to the Michealis-Menton equation with the values K1/2 = 1.99 ± 0.45 nM (n = 3) and 13.7 ± 3.0 nM (n = 3). Thus the K1/2 for Ca2+ pump activation at plasma divalent concentrations is 13.7 nM or pK = 7.86. Experiments in the absence of divalents were performed on different days from those in the presence of divalents.

Inhibition by Cd2+. The K1/2 for H+ activation of Ca2+ efflux could reflect a pK of an amino acid side chain of ~8.7 and would be consistent with titration of cysteine, histidine, lysine, or tyrosine. Cd2+ has the same charge and is almost the same size as Ca2+; it also binds better to cysteine or histidine than Ca2+. We found that the IC50 for extracellular Cd2+ inhibition was ~20 times lower than for Ca2+ at pH 7.2 (Fig. 4) in terms of total Cd2+ or Ca2+ in solution. Because Cd2+ complexes with Cl- with pK of ~1.5 (30), the free Cd2+ is probably <FR><NU>1</NU><DE>5</DE></FR> of the total Cd2+.


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Fig. 4.   Cd2+ inhibition of Ca2+ efflux is altered by extracellular H+ concentration. Cells were treated with DIDS so that the intracellular pH was clamped, and only the extracellular pH varied. Results of 2 separate experiments are shown. The IC50 values were 1.54 ± 0.27 mM (pH 7.2 with HEPES as the buffer, open circle ) and 0.35 ± 0.26 mM (pH 8.1 with TAPS as the buffer, triangle ).

If Cd2+ goes to the same site as Ca2+ and H+, then Cd2+ should inhibit better at lower H+ concentrations. This prediction is confirmed in Fig. 4; the decrease in IC50 with the decrease in H+ is consistent with a K1/2 for H+ activation of ~2 nM. Cd2+ may also bind to OH-; thus, the free concentration is a smaller fraction of the total at pH 8.1 than at pH 7.2, and the change in IC50 for total Cd2+ would underestimate the effect of H+/OH- on the IC50 for free Cd2+.

We determined that the Cd2+ inhibition was due to extracellular Cd2+ and not to Cd2+ entry and subsequent intracellular Cd2+ inhibition. Because intracellular Cd2+ is expected to remain trapped inside red blood cells, we measured the effect of reducing extracellular Cd2+ on Ca2+ efflux. If intracellular Cd2+ were the cause of the inhibition, removal of extracellular Cd2+ should have no effect. In contrast, Fig. 5 shows that reducing extracellular Cd2+ by adding the impermeant chelator EGTA resulted in a faster Ca2+ efflux, as expected, if the Cd2+ inhibition was a direct effect of extracellular Cd2+. Because there is no known mechanism for Cd2+ efflux from human red blood cells, this argues that the inhibition is due to extracellular Cd2+.


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Fig. 5.   Extracellular EGTA relieves the inhibition of Cd2+, showing that Cd2+ inhibition is extracellular. The cellular content of 45Ca2+ is plotted as a function of time. , Control efflux in the absence of Cd2+; open circle  and triangle , efflux in the presence of 10 mM Cd2+; open symbols are samples taken before 0.6 min. Between 0.6 and 0.8 min, EGTA was added and samples diluted. Final EGTA concentration was 2.63 mM, and final Cd2+ concentration was 2.32 mM. black-triangle, samples taken after 0.8 min. Efflux after reduction of free extracellular Cd2+ is similar to control cells not exposed to Cd2+.

Effect of MTSEA and MTSET. The higher affinity for Cd2+ is consistent with cysteine or histidine residues forming part of the Cd2+ inhibitory site that we feel is likely to be the Ca2+ release site. We found that the cysteine reagent MTSEA inhibited the pump when added to the extracellular media of intact red blood cells (Fig. 6). However, we found that incubation with MTSEA also modified intracellular glutathione so that we cannot determine whether the inhibition is due to modification of extracellular or intracellular cysteines. MTSET is a similar positive thiol reagent with a terminal triethylammonium group that is bulkier and that has a higher pK than the terminal amino group of MTSEA. MTSET did not inhibit Ca2+ efflux from intact cells (Fig. 6). In separate experiments, we did find that MTSET inhibits the Ca2+-ATPase activity of open membranes (data not shown). A simple explanation of the data is that histidine (and not cysteine) is present at the Cd2+ inhibitory site and is responsible for the higher affinity for Cd2+ inhibition compared with Ca2+. We plan to devise a strategy to convincingly modify extracellular histidines in future work.


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Fig. 6.   2-Aminoethyl methanethiosulfonate (MTSEA), but not 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), inhibits Ca2+ efflux. In other experiments, we have found that MTSEA, under these conditions, crosses the membranes, as it reduces the amount of 5,5'-dithio-bis(2-nitrobenzoate)-reactive groups in the red blood cell cytosol (see METHODS). Also, in other experiments, we have found that MTSET does inhibit the Ca2+-ATPase activity in open membranes. For MTSET n = 2 and for MTSEA n = 5. MTSEA has proved to be a useful control for determining if A-23187 is completely washed out during the 45Ca2+ loading procedure.

Note that the MTSEA inhibition of the PMCA provides a convenient method for determining that 45Ca2+ efflux is mediated by the PMCA and not by the Ca2+ ionophore A-23187.

Inhibition by Ni2+. Ni2+ also binds better to histidine residues than Ca2+. As shown in Fig. 7, Ni2+ inhibits better than Ca2+ but not as well as Cd2+. This is consistent with histidine as part of the release site. A summary of the IC50 values for Cd2+, Ni2+, and Ca2+ is presented in Table 1. Clearly the order of potency is Cd2+>Ni2+>Ca2+.


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Fig. 7.   Extracellular Ni2+ is a moderate inhibitor of the Ca2+ pump. Results of 3 separate experiments are shown. The IC50 was 11.3 ± 1.4 mM.


                              
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Table 1.   IC50 values for extracellular inhibition of Ca2+ efflux


    DISCUSSION
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INTRODUCTION
METHODS
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DISCUSSION
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In this paper, we have characterized extracellular properties of the PMCA and tested a critical prediction of the access well model. We have found that extracellular H+ activate with a pK of 8.7, that the IC50 for Ca2+ and Cd2+ decreases as H+ is decreased, consistent with Cd2+, Ca2+, and H+ competition, that the order of divalent cation IC50 values is Cd2+<Ni2+<Ca2+, which is consistent with the presence of either a cysteine or a histidine in the binding site, and that the cysteine reagent MTSET does not inhibit. The lack of effect of Em on the IC50 for Ca2+ inhibition is contrary to the predictions of a simple access well model.

Access well model and Em effects. Gassner et al. (10) studied the Em dependence of the Ca2+ pump in red blood cells. They found that the Ca2+ pump was slightly potential sensitive, as the flux changed ~10% for an estimated 100-mV change in Em. Although this is an important study, the authors did not attempt to maintain intracellular pH constant in intact cells. In separate experiments in inside-out vesicles and with open membranes, they suggest that changes of pH would not alter their findings. Xu and Roufogalis (35) studied the Em dependence of the Ca2+ pump in pH-clamped red blood cells and saw no change. Hao et al. (11) observed no increase in Ca2+ flux when the Em was collapsed in reconstituted proteoliposomes. These results are similar to those obtained on the Na+ pump in the absence of extracellular Na+ and the presence of saturating K+ (27, 29). (The comparable conditions for the Ca2+ pump are in the absence of extracellular Ca2+ and saturating extracellular H+.) These results indicate that, under these conditions, the rate-limiting step is not potential dependent. This can be achieved if a transport step (say, for Ca2+) is rate limiting and the transport step for the other ion (in this case H+) is potential dependent. Alternatively, because there is no extracellular product and the extracellular substrate is saturating, a high-field access well model will also give these results.

Three high-field access well models will be discussed (Fig. 8). In the first model, we assume that Ca2+ but not H+ must transverse an access well. In this case, the IC50 for Ca2+ inhibition is given by the equation
IC<SUB>50</SUB> = IC<SUB>50</SUB>(<IT>E</IT><SUB>m</SUB> = 0) × exp(−2 ⋅ &dgr; ⋅ <IT>E</IT><SUB>m</SUB> ⋅ <IT>F</IT>/<IT>RT</IT>)
where delta  is the fraction of the electrical field drop, and R, T, and F have their usual meanings (27). In the second model, we assume that Ca2+ binds in an access well and that one H+ binds in an access well that, for simplicity, involves the same electrical distance. Furthermore the one H+ and the Ca2+ are mutually exclusive. For this model
IC<SUB>50</SUB> = IC<SUB>50</SUB>(<IT>E</IT><SUB>m</SUB> = 0, H = 0)

× {[<IT>K</IT><SUB>½</SUB> + [H<SUP>+</SUP>] exp(1 ⋅ &dgr; ⋅ <IT>E</IT><SUB>m</SUB> ⋅ <IT>F</IT>/<IT>RT</IT>)]/<IT>K</IT><SUB>½</SUB>}

× exp(−2 ⋅ &dgr; ⋅ <IT>E</IT><SUB>m</SUB> ⋅ <IT>F</IT>/<IT>RT</IT>)
where K1/2 is the K1/2 for H+ and [H+] is the H+ concentration. The curves are plotted in Fig. 9 for a 54-mV change of Em (the smallest measured change of Em for the 3 experiments shown in Fig. 2A; with H+ = 12 nM and K+ = 2 nM). In these experiments, we made Em approx  EK by adding valinomycin and activating Ca2+-dependent K+ channels via Ca2+ loading (i.e., Gardos effect; see Ref. 22). Additionally, DIDS inhibits about one-half of the Cl- conductance in red blood cells (15). For the combined fit to the three experiments in Fig. 2B, the ratio of the IC50 values was 0.87, which is shown as the dashed line in Fig. 9. The lower dotted line is the ratio for two SD in the IC50 values. We believe that this intersection with the plot of ratio vs. delta  represents a reasonable upper bound for delta . Our data therefore argue against models in which Ca2+ must transverse a substantial amount of the membrane field to bind at the release site.


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Fig. 8.   Three high-field access well models for Ca2+ release from plasma membrane Ca2+ pump (PMCA). In A, Ca2+ must pass through a high-field access channel, but the conformation to which H+ binds does not have a high-field access channel. In B, both Ca2+ and H+ must pass through a high-field access channel to bind at the transport site. As discussed in the text, our data suggest that, if there is a high-field access channel, the fraction of the electrical field drop in the well (delta well) is <0.2. In C, there is a structural high-field access channel, but Ca2+ is not released through this channel and is released after the conformational change that results in a low-field access channel. This model is analogous to the Na+ pump high-field access channel. Because the Na+ pump transports 3 Na+ and the Ca2+ pump transports 1 Ca2+, there is a choice of whether the Ca2+ behaves more like the first Na+ (A) or more like the last 2 Na+ (C). delta T, fraction of electric field through which translocation step takes place.



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Fig. 9.   Changes in IC50 expected for different values of delta well, a measure of the electrical distance in an access well. Two different models are considered for a 54-mV change of Em. triangle , model in which only Ca2+ binding occurs in the access well; open circle , model in which both Ca2+ and 1 H+ bind in an access well. The ratio of the IC50 values for the combined fits was 0.87 (dashed line), and the ratio of 2 SD from the average was 0.73 (dotted line).

There is strong electrophysiological evidence for an access well for the Na+ pump (3, 9, 12, 23, 26, 27, 29). The Na+ pump and the Ca2+ pump are in the same family. Thus we were surprised that we did not observe a functional access channel. It is possible to maintain that the PMCA does have a structural ion well but that it is not observed functionally. This third access well model is shown in Fig. 8C. In both the Na+ pump model and in the model shown in Fig. 8C, there is a high-field access well. In the Na+ pump model, the first Na+ is released through this channel. In the Ca2+ pump model, there is no ion released through this channel. In both models, a conformational change then takes place so that now the access to the release sites is through a low-field access channel. In both the Na+ pump model and the Ca2+ pump model, the remaining ions (the last 2 Na+ and the only Ca2+) are released through this low-field access channel.

Histidine or cysteine in release site? We have examined several properties of the extracellular release site that suggest that a histidine or a cysteine may be important. The pK of H+ activation is consistent with histidine or cysteine (as well as tyrosine and lysine). It should be noted that the K1/2 for activation, in general, is a complex function of many rate constants of the Ca2+ pump cycle and may not necessarily provide a direct measure of the dissociation constant for H+ binding. In addition, the local environment of an amino acid may drastically change the pK for the side group.

Cd2+ and Ca2+ have similar sizes. The fact that Cd2+ binds better than Ca2+ is consistent with the presence of sulfur or nitrogen contributions to the binding site; the most obvious choices are cysteine and histidine. Even though cysteine binds Ni2+ and Cd2+ about equally well, whereas histidine binds Cd2+ better than Ni2+ (30), it is difficult to conclude from the different IC50 values for Cd2+ and Ni2+ that the release site contains histidine. This is because the geometry of the release site may also influence selectivity. Thus the difference in IC50 values between Ni2+ and Cd2+ may reflect their size difference.

MTSET, a reagent that can modify cysteines, did not inhibit Ca2+ efflux. Thus we favor the notion that there is a histidine in the Ca2+ release site. The smaller cysteine reagent, MTSEA, did inhibit from the outside; under our conditions, MTSEA was membrane permeant in red blood cells. MTSEA has also been shown to be permeant in excitable membranes (13). Because intracellular cysteine reagents have been shown to inhibit the Ca2+ pump (24), we have no reason to postulate a transmembrane cysteine accessible to MTSEA. However, we cannot rule out the possibility that there is an extracellular or transmembrane cysteine accessible to Cd2+ and Ni2+ (and perhaps MTSEA) but inaccessible to MTSET that is important in the Ca2+ pump cycle.

The current topological model of the PMCA has eight cysteines and three histidines in locations that would be consistent with being part of the Ca2+ release site and H+ transport site, i.e., in transmembrane domains or in the extracellular loops. These are listed in Table 2. All three histidines are conserved in all eight mammalian PMCA isoforms currently in GenBank (human PMCA 1-4, rat PMCA 1 and 2, rabbit PMCA 1, and pig PMCA 1). Some of the cysteines are conserved, some are not conserved, and none are conserved in regions with homology to two conformationally sensitive cysteines in the Na+ pump (17).

                              
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Table 2.   Locations of TM and extracellular cysteines and histidines

Extracellular H+ and divalent cation effects. As the Ca2+ pump mediates Ca2+/H+ exchange, extracellular H+ are a substrate for the Ca2+ pump. We and others have previously found that the Ca2+ pump is only slightly changed as H+ concentration is decreased from 100 (pH 7.0) to 10 (16, 19, 35) nM. In this study, we have extended the range of H+ concentrations examined. Because we use DIDS-treated red blood cells, the intracellular pH is clamped under these conditions. In this way, we were able to obtain the K1/2 of 2 nM for extracellular H+ activation.

Both Kratje et al. (16) and Xu and Roufogalis (35) have studied the inhibition of the PMCA by extracellular Ca2+. They obtained IC50 values of 11 and 6 mM, respectively. Our value is slightly higher, and this probably reflects the difference in pH in the studies.

Kratje et al. (16) showed that extracellular Ca2+ inhibition was more potent at alkaline pH using resealed red blood cell ghosts; however, the pH was varied intracellularly and extracellularly (16). In our study, the intracellular pH was clamped, and only the extracellular pH was varied; our results are strikingly similar, suggesting that, under their conditions, most of the inhibition was mediated by extracellular H+. If the primary effect of H+ in this pH range is at the transport site, this is not surprising because the intracellular Ca2+ concentration (at pH 7) was ~100-fold greater than the K1/2 for Ca2+, and thus it is plausible that the intracellular site remained saturated with Ca2+ as the pH was decreased. Xu and Roufogalis (35) also showed that extracellular Ca2+ became more potent at alkaline pH using the A-23187 and cobalt technique. Our study confirms their results and proves more quantitative data on the interaction between Ca2+ and H+. It is satisfying that these two separate techniques give similar answers. The Ca2+ pump in squid axons has been shown to have similar interactions between extracellular Ca2+ and extracellular H+ (4).

Our IC50 for extracellular Cd2+ inhibition at pH 7.2 agrees well with the value obtained by Xu and Roufogalis (35) using the A-23187 and cobalt technique. The fact that extracellular H+ have the same effect on the IC50 for Cd2+ as for Ca2+ argues that H+, Ca2+, and Cd2+ are competing at a similar site, although one can construct more complicated models with separate sites that will also give this result.

From the pH dependence of the IC50 for Ca2+, we can determine the inhibition constant for Ca2+ in the absence of H+. The values range from 0.5 to 1.0 mM. Similar values are obtained from the increase in K1/2 for H+ activation by the addition of 1 mM Ca2+ and 1 mM Mg2+.

Physiological significance. The activation by H+ (K1/2 = 2 nM) and the inhibition by Ca2+ (IC50 = 10 mM at pH 7.4) have kinetic constants that are far from the normal physiological values (H+ = 40 nM, Ca2+ = 1 mM). Thus at pH 7.4 (40 nM) the pump is essentially saturated in the absence of Ca2+; however, at physiological Ca2+ and Mg2+ values (~1 mM free, each), we estimate that the pump is only at 90% of the maximum, a figure that agrees with the value obtained previously (16). In this regard, the PMCA is similar to the Na+ pump, which has high affinity for K+ in the absence of Na+, but, at physiological concentrations, K+ is nearly but not completely saturating.

Number of H+. The H+ activation curves in Figs. 1 and 3 are both fit with a hyperbolic curve, i.e., a Hill coefficient of 1. The stoichiometry of PMCA has been reported to be 1 Ca2+-2 H+ and 1 Ca2+-1 H+ (11, 19, 21, 25, 26, 31), and our activation curves are consistent with 1 H+ activating. However, although our data do not require a model with 2 H+ activating, our data also do not eliminate such a model. K+ activation of the Na+ pump activation is hyperbolic in the absence of extracellular Na+, yet current models all have the stoichiometry as 2 K+, so n = 1 does not eliminate n = 2 models.

In this paper, we have characterized extracellular properties of the PMCA and tested a critical prediction of the access well model. We have found that extracellular H+ activate with a pK of 9, that the change of IC50 for Ca2+ and Cd2+ as pH is altered is consistent with Ca2+ and H+ competition, that the order of divalent cation IC50 values is Cd2+<Ni2+<Ca2+, which is consistent with the presence of either a cysteine or a histidine in the binding site, and that the cysteine reagent MTSET does not inhibit. The lack of effect of Em on the IC50 for Ca2+ inhibition is contrary to the predictions of a simple access well model.


    ACKNOWLEDGEMENTS

We thank Dr. Craig Gatto for very helpful discussions on the manuscript.


    FOOTNOTES

Financial support for this work was provided by National Institutes of Health Grants DK-37512 (M. A. Milanick) and HL-07094 (L. Huang).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: M. Milanick, Dept. of Physiology, MA415 Medical Sciences Bldg., Univ. of Missouri, Columbia, MO 65212 (E-mail: milanickm{at}missouri.edu).

Received 13 May 1999; accepted in final form 30 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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