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Department of Physiology and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri 65212
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
/HCO3 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.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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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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
~
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).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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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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 of the total Cd2+.
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; 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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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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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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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
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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
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![× {[<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>}](/content/vol278/issue5/fulltext/C965/img004.gif)
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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. represents a reasonable upper bound for . 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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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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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 |
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We thank Dr. Craig Gatto for very helpful discussions on the manuscript.
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FOOTNOTES |
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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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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)
with K+ (
) 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.
, Control efflux in the absence of Cd2+; 
, samples taken after
0.8 min. Efflux after reduction of free extracellular Cd2+
is similar to control cells not exposed to
Cd2+.
