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
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ABSTRACT |
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 |
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.
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METHODS |
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+ ( )
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.
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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 |
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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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.
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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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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, ) and 0.35 ± 0.26 mM (pH 8.1 with TAPS as the buffer,
).
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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+; and ,
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. , samples taken after
0.8 min. Efflux after reduction of free extracellular Cd2+
is similar to control cells not exposed to
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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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.
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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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DISCUSSION |
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
where
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
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
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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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 ( 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). 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
well, a measure of the electrical distance in an
access well. Two different models are considered for a 54-mV change of
Em. , model in which only Ca2+
binding occurs in the access well; , 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).
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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).
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 |
1.
Carafoli, E,
and
Stauffer T.
The plasma membrane calcium pump: functional domains, regulation of the activity, and tissue specificity of isoform expression.
J Neurobiol
25:
312-324,
1994[ISI][Medline].
2.
Daugirdas, JT,
Arrieta J,
Ye M,
Flores G,
and
Battle DC.
Intracellular acidification associated with changes in free cytosolic calcium. Evidence for Ca2+/H+ exchange via a plasma membrane Ca2+(2+)-ATPase in vascular smooth muscle cells.
J Clin Invest
95:
1480-1489,
1995[ISI][Medline].
3.
DeWeer, P,
Rakowski RF,
and
Gadsby DC.
Voltage sensitivity of the Na/K pump: structural implicatons. The Sodium Pump, edited by Bamberg E,
and Schoner W.. New York: Springer, 1994, p. 472-481.
4.
Dipolo, R,
and
Beauge L.
The effect of pH on Ca2+ extrusion mechanisms in dialyzed squid axons.
Biochim Biophys Acta
688:
237-245,
1982[ISI][Medline].
5.
Dissing, S,
and
Hoffman JF.
Ouabain-insensitive Na efflux from human red blood cells stimulated by outside H, Na or Li ions (Abstract).
J Gen Physiol
80:
15a,
1982.
6.
Forgac, M,
and
Cantley L.
The plasma membrane (Mg2+)-dependent adenosine triphosphatase from the human erythrocyte is not an ion pump.
J Membr Biol
80:
185-190,
1984[ISI][Medline].
7.
Freedman, JC,
and
Novak TS.
Use of triphenylmethylphosphonium to measure membrane potentials in red blood cells.
Methods Enzymol
173:
94-100,
1989[ISI][Medline].
8.
Furuya, H,
Jacobson HR,
and
Breyer MD.
Evidence for basolateral membrane Ca2+/H+ exchange in outer medullary collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F88-F93,
1993[Abstract/Free Full Text].
9.
Gadsby, DC,
Rakowski RF,
and
De Weer P.
Extracellular access to the Na,K pump: pathway similar to ion channel.
Science
260:
100-103,
1993[ISI][Medline].
10.
Gassner, B,
Luterbacher L,
Schatzmann HJ,
and
Wuthrich A.
Dependence of the red blood cell calcium pump on the membrane potential (Abstract).
Cell Calcium
9:
95,
1988[ISI][Medline].
11.
Hao, L,
Rigaud J-L,
and
Inesi G.
Ca2+/H+ countertransport and electrogenicity in proteoliposomes containing erythrocyte plasma membrane Ca2+-ATPase and exogenous lipids.
J Biol Chem
269:
14268-14275,
1994[Abstract/Free Full Text].
12.
Hilgemann, DW.
Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches.
Science
263:
1429-1432,
1994[ISI][Medline].
13.
Holmgren, M,
Liu Y,
Xu Y,
and
Yellen G.
On the use of thiol-modifying agents to determine channel topology.
Neuropharmacology
35:
797-804,
1996[ISI][Medline].
14.
Jennings, ML.
Structure and function of the red blood cell anion transport protein.
Annu Rev Biophys Biophys Chem
18:
397-430,
1989[ISI][Medline].
15.
Knauf, PA,
Law FY,
and
Marchant PJ.
Relationship of net chloride flow across the human erythrocyte membrane to the anion exchange mechanism.
J Gen Physiol
81:
95-126,
1983[Abstract].
16.
Kratje, RB,
Garrahan PJ,
and
Rega AF.
Two modes of inhibition of the Ca2+ pump in red cells by Ca2+.
Biochim Biophys Acta
816:
365-378,
1985[ISI][Medline].
17.
Lutsenko, S,
Daoud S,
and
Kaplan JH.
Identification of two conformationally sensitive cysteine residues at the extracellular surface of the Na,K-ATPase alpha-subunit.
J Biol Chem
272:
5249-5255,
1997[Abstract/Free Full Text].
18.
Lutsenko, S,
and
Kaplan JH.
P-type ATPases (Abstract).
Trends Biochem Sci
21:
467,
1996[ISI][Medline].
19.
Milanick, MA.
Proton fluxes associated with the Ca2+ pump in human red blood cells.
Am J Physiol Cell Physiol
258:
C552-C562,
1990[Abstract/Free Full Text].
20.
Milanick, A,
and
Hoffman M.
Separate effects of internal JF and external pH on cation influxes in human red blood cells studied by means of a pH clamp (Abstract).
J Gen Physiol
80:
20a,
1982.
21.
Niggli, V,
Sigel E,
and
Carafoli E.
The purified Ca2+ pump of human erythrocyte membranes catalyzes an electroneutral Ca2+-H+ exchange in reconstituted liposomal systems.
J Biol Chem
257:
2350-2356,
1982[Free Full Text].
22.
Parker, JC,
and
Dunham PB.
Passive cation transport.
Hematology
11:
507-561,
1989.
23.
Rakowski, RF,
Gadsby DC,
and
DeWeer P.
Voltage dependence of the Na/K pump.
J Membr Biol
155:
105-112,
1997[ISI][Medline].
24.
Richards, DE,
Rega AF,
and
Garrahan PJ.
ATPase and phosphatase activities from human red cell membranes. I. The effects of N-ethylmaleimide.
J Membr Biol
35:
113-124,
1977[ISI][Medline].
25.
Romero, JP,
and
Ortiz CEP
Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes.
J Membr Biol
101:
237-246,
1988[ISI][Medline].
26.
Rossi, PJ,
and
Schatzmann HJ.
Is the red cell calcium pump electrogenic?
J Physiol (Lond)
327:
1-15,
1982[Abstract].
27.
Sagar, A,
and
Rakowski RF.
Access channel model for the voltage dependence of the forward-running Na+/K+ pump.
J Gen Physiol
103:
869-893,
1994[Abstract].
28.
Salvador, JM,
Inesi G,
Rigaud JL,
and
Mata AM.
Ca2+ transport by reconstituted synaptosomal ATPase is associated with H+ countertransport and net charge displacement.
J Biol Chem
273:
18230-18234,
1998[Abstract/Free Full Text].
29.
Schwartz, W,
Vasilets LA,
Omay H,
Efthymiadis E,
Rettinger J,
and
Elsner S.
Electrogenic properties of the endogenous and of modified Torpedo Na/K pumps in Xenopus oocytes: the access channel for external cations.
In: The Sodium Pump, edited by Bamberg E,
and Schoner W.. New York: Springer, 1994, p. 482-494.
30.
Sillen, GL,
and
Martell AE.
Stability Constants of Metal-Ion Complexes. London: The Chemical Society, 1971, p. 180, 308, 450.
31.
Smallwood, JI,
Waisman DM,
Lafreniere D,
and
Rasmussen H.
Evidence that the erythrocyte calcium pump catalyzes a Ca2+:H+ exchange.
J Biol Chem
258:
11092-11097,
1983[Abstract/Free Full Text].
32.
Wagg, J,
Holmgren M,
Gadsby DD,
Benzanilla F,
Rawkowski RF,
and
De Weer P.
Na/K pump-mediated deocclusion steps accompanying extracellular release of three sodium ions.
J Gen Physiol
108:
30a-31a,
1996.
33.
Xu, W,
and
Milanick MA.
The plasma membrane Ca pump, (PMCA) flux in human erythrocytes is membrane potential dependent (Abstract).
Biophys J
70:
A329,
1996.
34.
Xu, W,
Parkinson EL,
Wilson BJ,
and
Milanick MA.
Inhibition of the plasma membrane calcium pump (Abstract).
Biophys J
76:
A379,
1999[ISI].
35.
Xu, YH,
and
Roufogalis BD.
Asymmetric effects of divalent cations and protons on active Ca efflux and Ca-ATPase in intact red blood cells.
J Membr Biol
105:
155-164,
1988[ISI][Medline].
Am J Physiol Cell Physiol 278(5):C965-C972
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