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INTRODUCTION |
As it grows and matures within its host erythrocyte, the
intracellular malaria parasite takes up nutrients from the external medium, doing so in competition with the cells of the host. The mechanisms by which it does so remain poorly understood. One important nutrient for which the human parasite P. falciparum has an
absolute extracellular requirement is the vitamin pantothenic acid
(PA1, vitamin B5)
(1), a precursor of the enzyme cofactor coenzyme A (CoA) (2). PA is
impermeant to normal, uninfected human erythrocytes but enters P. falciparum-infected erythrocytes via "new permeation pathways"
induced by the intracellular parasite in the host cell membrane (3).
Once inside the parasitized cell, PA is taken up by the parasite where
it is phosphorylated by pantothenate kinase (3), the first step in its
conversion to CoA.
The aim of this study was to investigate the mechanism by which PA is
taken up across the plasma membrane of the intracellular malaria
parasite. The results demonstrate the presence in the parasite membrane
of a low affinity H+:pantothenate symporter, which acts in
combination with a high affinity kinase within the parasite to form a
highly efficient uptake system. The characteristics of the
H+-dependent pantothenate transporter are quite
different from those of its Na+-dependent
counterparts in mammalian cells, raising the possibility that it may be
a suitable antimalarial drug target.
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EXPERIMENTAL PROCEDURES |
Parasite Culture and Isolation
P. falciparum parasites (FAF6, cloned from ITG2, (4))
were maintained in synchronous in vitro cultures as
described previously (5), with modifications (6). All experiments were
carried out on late-stage trophozoites (36-40 h post-invasion)
isolated from their host cells using saponin (0.05% w/v) as described
previously (3, 7). Saponin permeabilizes both the host cell membrane and the parasitophorous vacuole membrane (8), thereby allowing solutes
present in the extracellular medium unimpeded access to the parasite
plasma membrane.
[14C]PA Uptake Measurements
Uptake into Isolated Parasites--
Measurements of the uptake
of [14C]PA into parasites within saponin-permeabilized
erythrocytes were carried out as described previously (3). In
experiments designed to allow investigation of the uptake of
[14C]PA under conditions in which the phosphorylation of
the radiolabeled compound within the cell was minimized, isolated
parasites were depleted of ATP by suspending them in a glucose-free,
HEPES-buffered saline (135 mM NaCl, 5 mM KCl,
25 mM HEPES, 1 mM MgCl2, pH 7.4) for 30-60 min at 37 °C (7, 9). The ATP-depleted parasites were then
harvested by centrifugation (2060 × g, 8 min) and
resuspended in a weakly buffered solution (135 mM NaCl, 5 mM KCl, 0.1 mM HEPES, 1 mM
MgCl2, pH 7.4) at 0.6-1.7 × 109
cells/ml, before combining 1 volume of the ATP-depleted parasite suspension with 12 volumes of a heavily buffered solution (135 mM NaCl, 5 mM KCl, 25 mM HEPES, 20 mM MES, 1 mM MgCl2), pH-adjusted to
either 5.5 or 7.4 and containing [14C]PA (± inhibitors),
to give a final PA concentration of 2 µM and a
[14C]PA activity of 0.1 µCi/ml. At predetermined
intervals 0.2-ml aliquots of the suspension were removed and
centrifuged through an oil layer to terminate the flux as described
elsewhere (3, 7).
Rat Hepatoma Cells--
In one series of experiments the ion
dependence of [14C]PA uptake into isolated parasites was
compared with that of [14C]PA uptake into mammalian cells
(the rat hepatoma cell line, HTC). The HTC cells were grown as adherent
cultures under standard conditions and were released into suspension
(to allow the use of identical methodology to that used with the
isolated parasites) by trypsinization. Uptake of [14C]PA
into HTC cells in suspension was carried out using the same solutions
and under identical conditions to those used for the isolated parasites.
Intracellular Water Volume Measurements
The water volume of the cell pellets (both for isolated
parasites and HTC cells) was estimated using a combination of
[3H]water and [14C]sucrose as described
elsewhere (7). The water volumes of the isolated parasites and HTC
cells used in this study were estimated as 20 ± 2 fl (mean ± S.E.; n = 13) and 67 ± 1 fl (mean ± range/2; n = 2), respectively.
Phosphorylation of [14C]PA by Parasite Lysates
Phosphorylation of [14C]PA by cell lysates
prepared from isolated parasites was monitored as described previously
(3). Cell lysates were either used immediately or frozen and stored at
70 °C for subsequent analysis. It was confirmed in control
experiments that no loss of activity occurred during cryostorage.
Cytosolic pH Measurements and Estimation of the Cytosolic
Buffering Capacity
The cytosolic pH (pHi) of the isolated parasites was
monitored using the intracellular fluorescent pH indicator BCECF, as
described previously (7). Measurements were carried out at 37 °C
(except where specified otherwise) using a PerkinElmer Life Sciences
LS-50B spectrofluorometer, with gentle stirring of the cell suspension.
The buffering capacity of the parasite cytosol (
i) was
measured both at 37 °C and 5 °C by the additions of varying
concentrations (10-40 mM) of either
NH
or butyrate to isolated parasites
suspended in either the presence or absence of glucose. The removal of
glucose results in a decrease in pHi of about 0.2 pH units (7)
and served the purpose of increasing the pHi range over which
i could be estimated. The discrepancy between the change
in pHi predicted to occur upon addition of different
concentrations of either NH
or
butyrate, and that actually observed, allows the calculation of
i (10).
For parasites at 37 °C,
i decreased approximately
linearly with pHi, from ~30 mmol of H+/(l cell
H2O.pH unit) at pHi 7.5, to ~50 mmol of
H+/(l cell H2O.pH unit) at pHi 7.1. At
pHi values below 7.1,
i increased more steeply with
decreasing pHi, reaching a value of ~150 mmol of
H+/(l cell H2O.pH unit) at a pHi of 6.9. Linear regression analysis of the pHi dependence of
i in the pHi range 7.5-7.1 yielded an estimated
i value of 42.2 mmol of H+/(l cell
H2O.pH unit) at the normal resting pHi of 7.3. This
value was used in the evaluation of the H+ transport rates
in all experiments carried out at 37 °C.
For parasites at 5 °C,
i remained at ~30 mmol of
H+/(l·pH unit) over the pHi range 7.5-7.1, and
increased only marginally to ~50 mmol of H+/(l cell
H2O.pH unit) when the pHi was decreased further to 7.0. The
i value at the resting pHi at
5 °C (7.22 ± 0.02; mean ± S.E.; n = 4) was
estimated to be 32.3 mmol of H+/(l cell H2O.pH unit).
Because the
i values at the resting pHi values at
37 °C and 5 °C were similar, an average
i value of
37.3 mmol of H+/(l cell H2O.pH unit) was used for
the estimation of H+ influx rates in the analysis of the
experiment (giving rise to Fig. 6) in which H+ influx was
measured as a function of temperature in the range 16-37 °C.
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RESULTS |
PA Uptake by P. falciparum Trophozoites Is
Na+-independent and pH-dependent--
Uptake
of [14C]PA into isolated P. falciparum
trophozoites was independent of the presence of Na+ in the
extracellular solution. The initial rate of accumulation of
[14C]PA in parasites suspended in a HEPES-buffered saline
solution, estimated from the initial slope of the uptake time course
(Fig. 1A), was 2.8 ± 0.4 µmol/(1012 parasites.h) (mean ± S.E.;
n = 4). This was not significantly different from that
in parasites suspended in Na+-free solution, containing
choline in place of Na+ (2.7 ± 0.4 µmol/(1012 parasites.h); n = 4;
p = 0.2, paired t test).

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Fig. 1.
Effect of extracellular Na+ and
pH on the accumulation of [14C]PA by isolated P. falciparum trophozoites. A, time courses for
the accumulation of [14C]PA (2 µM) by
isolated parasites suspended in either HEPES-buffered saline
(containing 125 mM NaCl; closed circles) or a
Na+-free solution containing choline in place of
Na+ (open circles). The data are averaged from
four separate experiments, each carried out in duplicate, and are
shown ± S.E. B, time courses for the accumulation of
[14C]PA by rat hepatoma (HTC) cells suspended in the
Na+-containing (closed circles) or
Na+-free (open circles) media used in
A. The data are from a single experiment representative of
those obtained from two separate experiments. C, time
courses for the uptake of [14C]PA (2 µM) by
isolated parasites suspended in HEPES-buffered RPMI 1640 at pH 6.5 (closed diamonds), 7.4 (open squares), or 8.5 (open triangles). Data are averaged from two separate
experiments, each carried out in duplicate and are shown ± range/2.
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This contrasts with the situation in mammalian cells. As illustrated in
Fig. 1B, the uptake of [14C]PA into rat
hepatoma (HTC) cells was wholly dependent upon the presence of
Na+ in the extracellular medium, decreasing to a negligible
level on replacement of Na+ with choline.
Although independent of the presence of extracellular Na+,
the rate of [14C]PA uptake did show a marked dependence
on the extracellular pH (Fig. 1C). For isolated parasites
suspended in RPMI at pH 8.5 the initial rate of uptake was 0.5 ± 0.2 µmol/(1012 parasites.h). Decreasing the pH of the
medium (pHo) from 8.5 to 7.4 caused the initial rate of uptake
to increase significantly (p = 0.042) to 1.1 ± 0.2 µmol/(1012 parasites.h). Decreasing the pHo
further to 6.5 caused the initial rate of uptake to increase even
further (p = 0.009), to 1.7 ± 0.2 µmol/(1012 parasites.h).
Isolated trophozoites accumulated [14C]PA to levels
many-fold higher than that in the extracellular medium (Fig. 1,
A and C) and the data of Fig. 1C are
consistent with a role for H+ in the PA uptake process. As
has been shown previously (3), however, PA entering the parasite is
phosphorylated, the first step in its conversion to coenzyme A. The
accumulation of radiolabel under the conditions of Fig. 1 therefore
arises from a combination of the transport of the compound into the
parasites and its subsequent "metabolic trapping" within
the parasites (7).
PA Transport into ATP-depleted P. falciparum Trophozoites Is
H+-dependent and Inhibited by Phloretin and by
Sulfhydryl Reagents--
To investigate the possible role of
H+ ions in the transport of PA into the parasite, in the
absence of interference from metabolism, uptake of
[14C]PA was measured into parasites depleted of ATP (by
preincubation in a glucose-free solution at pH 7.4; see "Experimental
Procedures") and therefore unable to carry out the phosphorylation of
PA. The ATP-depleted parasites lose the ability to regulate their
cytosolic pH (pHi), which, over a period of several minutes,
therefore approaches the pH of the preincubation medium (7). Following ATP depletion, the cells were resuspended (at t = 0) in
media of either pH 5.5 or 7.4 (i.e. in either the presence
or absence, respectively, of a substantial initial inward
[H+] gradient).
In the absence of a significant inward [H+] gradient
(Fig. 2A, open
symbols) there was a gradual uptake of [14C]PA, with
the intracellular radiolabel reaching a concentration of around 0.8 times that in the extracellular solution by 5 min. By contrast, in
ATP-depleted parasites suspended in the presence of a substantial
inward [H+] gradient (Fig. 2A, closed
symbols), there was a rapid uptake of radiolabel into the
parasites. The ratio of the intracellular concentration of radiolabel
to the extracellular concentration of radiolabel (the so-called
"distribution ratio") reached a maximum value of 6-7 at 1-2 min,
before declining.

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Fig. 2.
H+-dependent uptake
of [14C]PA into ATP-depleted P. falciparum
trophozoites. A, time courses for the uptake of
[14C]PA (2 µM) into isolated parasites
depleted of ATP by preincubation in glucose-free media (pH 7.4) then
resuspended (at t = 0) in a HEPES- and MES-buffered
solution at a pHo of 7.4 (open circles), or 5.5 (closed circles). Data are averaged from three separate
experiments, each carried out in duplicate and are shown ± S.E.
Analysis of the cell extracts using Somogyi reagent (3) revealed that,
throughout the time course at pHo of 5.5, >80% of the
intracellular label remained non-phosphorylated. B, time
courses for the dissipation of the inward H+ gradient (as
indicated by the "BCECF fluorescence ratio" (i.e. 495 nm/440 nm)) in cells depleted of ATP by preincubation in glucose-free
medium at pH 7.4 then resuspended in medium of pH 5.5 at
t = 0, in the absence of PA and in the presence or
absence of the pharmacological agents used in C. The
ordinate shows the BCECF fluorescence ratio rather than
pHi as the dissipation of the H+ gradient caused
pHi to decrease to values outside the effective range of the
dye. C, effect of pharmacological agents on the
H+-induced [14C]PA (2 µM)
influx into ATP-depleted, saponin-permeabilized P. falciparum-infected erythrocytes. Uptake was measured over 1 min
in ATP-depleted cells suspended (at t = 0, at
concentrations of ~20 × 106 cells/ml) in
medium at a pH of either 5.5 or 7.4 (as indicated). The
inhibitors were added to the parasite suspension in combination with
the [14C]PA (i.e. there was no preincubation
of the cells with inhibitors). The data are averaged from four
separate experiments, each performed in duplicate, and are shown ± S.E. NEM, N-ethylmaleimide.
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Cell extracts corresponding to each of the time points shown by the
closed symbols in Fig. 2A were analyzed for the
presence of phosphorylated [14C]PA using Somogyi reagent
(3). Throughout the time course the majority (>80%) of the PA taken
up by the parasites remained unphosphorylated. The accumulation of
[14C]PA, within the ATP-depleted parasites, to
concentrations much higher than that in the extracellular medium, as
seen in parasites suspended in the presence (but not in the
absence) of an inward [H+] gradient, may therefore be
attributed primarily to active accumulation of (non-phosphorylated)
[14C]PA.
The inability of the ATP-depleted parasites to regulate their
pHi meant that those suspended in the acidic medium underwent a
progressive acidification (Fig. 2B). This accounts for the
observed decline in the PA distribution ratio following its reaching a
maximum at 1-2 min (Fig. 2A, closed
symbols).
Fig. 2C shows the effect of a number of reagents on the
uptake of [14C]PA by ATP-depleted parasites suspended in
the presence of an inward [H+] gradient, whereas Fig.
2B illustrates the effect of the different reagents tested
on the time-dependent dissipation of the [H+]
gradient. In this set of experiments, the distribution ratio in the
absence of inhibitors reached a value of ~4.5 in the 1-min incubation
period. The H+ ionophore carbonyl cyanide
m-chlorophenylhydrazone (CCCP) (10 µM)
caused a rapid dissipation of the imposed [H+] gradient
(Fig. 2C) and caused the distribution ratio attained in 1 min to decrease to just below 1, a similar value to that seen in cells
suspended in the absence of a transmembrane [H+] gradient
at pHo 7.4 (Fig. 2C). Phloretin (200 µM), and the two sulfhydryl reagents pCMBS (10 µM) and N-ethylmaleimide (2 mM), were all found to reduce the uptake of
[14C]PA by
70% (p
0.008; Fig.
2B). Unlike CCCP, however, these reagents did not
increase the rate of dissipation of the imposed [H+]
gradient (Fig. 2B) and their inhibitory effect on
[14C]PA uptake may therefore be attributed to an
inhibition of the actual transport mechanism rather than a dissipation
of the driving force for PA influx.
Pantothenate Influx into P. falciparum Trophozoites Is Accompanied
by H+ Influx--
The data of Fig. 2, demonstrating
H+-dependent PA transport, are consistent with
the hypothesis that pantothenate is transported across the parasite
plasma membrane via a H+-coupled process. This was explored
further by investigating the effect of pantothenate on the pHi
of the isolated parasites. As shown in Fig.
3A, the initial resting
pHi of the parasites at 37 °C was in the range 7.30-7.35.
Addition of pantothenate (2-40 mM) to the suspending
medium resulted in a transient, concentration-dependent acidification of the parasite cytosol, consistent with H+
entering the parasite. The time courses for the initial acidification were fitted to a first order exponential equation (Fig. 3B)
and the initial slope (i.e. the initial rate of
acidification of the cytosol) was calculated for each of the fitted
curves. This was multiplied by the intracellular buffering capacity
(42.2 mmol of H+/(l cell H2O.pH unit)) to give an
initial rate of H+ influx at each of the pantothenate
concentrations tested. Fig. 3C shows the initial
H+ influx rate as a function of the extracellular
pantothenate concentration. The data, showing a non-linear dependence
of H+ influx rate on pantothenate concentration, were
fitted to the Michaelis-Menten equation, giving an estimated
Km of 22.6 ± 2.3 mM and
Vmax of 56.3 ± 2.2 mmol of
H+/(l cell H2O.min) (mean ± S.E.;
n = 4). The water volume of a single isolated P. falciparum trophozoite estimated in this study was 20 fl
("Experimental Procedures"); the estimated
Vmax is therefore equivalent to a PA-induced
H+ influx of 1.13 ± 0.04 mmol of
H+/(1012 cells.min).

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Fig. 3.
Pantothenate-induced H+ influx in
isolated P. falciparum trophozoites suspended in a
solution of pH 7.1. A, effect of the addition (at the
points indicated by the filled arrowheads) of increasing
concentrations of pantothenate (pH 7.1) on the pHi of isolated
parasites. B, time-dependent change in
pHi in the initial period following the addition of increasing
concentrations of pantothenate (pH 7.1). pHi is the
difference between the resting pHi prior to the addition of
pantothenate (an average of the pHi values obtained over the
20 s immediately prior to the addition of pantothenate) and the
pHi at a given time (t) following pantothenate
addition. The data were fitted to the first order equation pH = pH × (1 e kt), where
pH is the maximum
decrease in pH following the addition of pantothenate and k
is the first order rate constant for the acidification. C,
concentration dependence of the initial rate of pantothenate-induced
H+ influx. The initial rate of pantothenate-induced
H+ influx at each concentration was estimated in each case
by the expression H+ influx = i × pH × k,
where i is the intracellular buffering capacity (42.2 mmol
of H+/(l cell H2O.pH unit)) and
pH and k
are from the fitted curves shown in B. The data were fitted
to the Michaelis-Menten equation (H+ influx = Vmax × [pantothenate]o/(Km + [pantothenate]o)). Km was estimated as
22.6 ± 2.3 mM and Vmax as
56.3 ± 2.2 mmol of H+/(l cell H2O.min)
(mean ± S.E.; n = 4). The data in A
are from a single experiment representative of those obtained in four
separate experiments. The data in B and C are
averaged from four separate experiments; error bars in
B have been omitted for clarity; error bars in
C represent S.E.
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Pantothenate-induced H+ Influx Is Inhibited by
Phloretin and pCMBS--
Phloretin and pCMBS, shown in Fig.
2C to inhibit H+-dependent
[14C]PA uptake, were both found to be effective
inhibitors of the pantothenate-induced acidification. The addition of
200 µM phloretin to ATP-replete parasites caused a slight
apparent alkalinization of the cytosol (by an undetermined mechanism)
and an almost complete inhibition of the acidification caused by the
addition of 40 mM PA (not shown). To eliminate the
possibility that the inhibition of H+ influx was related to
the apparent phloretin-induced alkalinization, the experiment was
repeated in parasites depleted of ATP, which caused pHi to
decrease from 7.3 to 7.1 (Fig.
4A and Ref. 7) and which
abolished the apparent phloretin-induced alkalinization. As shown in
Fig. 4A, 200 µM phloretin inhibited the
pantothenate-induced acidification in ATP-depleted parasites almost
completely. Fig. 4B shows the concentration dependence of
the inhibition by phloretin of the pantothenate-induced acidification;
the IC50 (i.e. the concentration of phloretin at
which inhibition was half-maximal) was 16.4 ± 2.6 µM (mean ± S.E.; n = 3; Fig.
4B).

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Fig. 4.
Effect of the transport inhibitors phloretin
and pCMBS on PA-induced H+ influx into
isolated P. falciparum trophozoites suspended in a
solution of pH 7.1. A, the effect of
Me2SO (upper trace) and phloretin (200 µM added in Me2SO; lower trace) on
the cytosolic acidification induced by the addition of 40 mM pantothenate (pH 7.1) to ATP-depleted isolated
parasites. The point of addition of the Me2SO/inhibitor is
indicated by the open arrowheads, and the point of addition
of the pantothenate is indicated by the closed arrowheads.
The data are from a single experiment, representative of those obtained
in three separate experiments. B, dose-response curve for
the effect of phloretin on the initial rate of pantothenate-induced
H+ influx. Data averaged from three separate experiments
(shown ± S.E.) were fitted by non-linear least squares regression
to a sigmoidal curve. C, the effect of water (upper
trace) and pCMBS (6.25 µM added in water;
lower trace) on the cytosolic acidification induced by the
addition of 40 mM pantothenate (pH 7.1). The point of
addition of the water/inhibitor is indicated by the open
arrowheads, and the point of addition of the pantothenate is
indicated by the closed arrowheads. The data are from a
single experiment, representative of those obtained in three separate
experiments. D, dose-response curve for the effect of
pCMBS on the initial rate of PA-induced H+
influx. Data are from a single experiment, representative of those
obtained in three separate experiments.
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The sulfhydryl reagent, pCMBS, was an even more potent
inhibitor of the pantothenate-induced acidification, exerting an
unusual "all-or-none" effect. In initial experiments it was found
that pCMBS concentrations as low as 0.3 µM
caused complete inhibition of the pantothenate-induced acidification
(Fig. 4C). However, it was also found that inhibition of the
pantothenate-induced acidification by pCMBS was dependent
upon the concentration of cells present in the suspension; 0.391 µM pCMBS abolished pantothenate-induced acidification in parasites suspended at a concentration of 4.3 × 106 cells/ml while having no effect on pantothenate-induced
acidification in parasites suspended at 8.6 × 106
cells/ml (not shown). Fig. 4D, showing the concentration
dependence of the effect of pCMBS on pantothenate-induced
acidification in a single representative experiment, illustrates the
all-or-none effect. Under the conditions of the experiment, 0.781 µM pCMBS caused a complete inhibition of
pantothenate-induced H+ influx whereas 0.391 µM pCMBS was without significant effect.
Pantothenate:H+ Transport Operates with a Stoichiometry
of 1:1 and Shows a Strong Temperature Dependence--
The data in
Figs. 2 and 3, together, constitute good evidence for PA entering the
parasite via a H+:pantothenate symport process. The
stoichiometry of the process was investigated by comparing the maximum
decrease in pHi (
pH
) observed
following the addition of increasing concentrations of pantothenate
(2.5-40 mM) with that observed following the addition of
the same concentrations of the more lipophilic monocarboxylate,
butyrate. Butyrate crosses membranes rapidly in the undissociated form;
i.e. with an effective stoichiometry of transport of 1 H+:1 butyrate (11). For the purpose of these experiments
the isolated parasites were depleted of ATP to minimize H+
pump-mediated pH recovery within the time taken for the pantothenate- and butyrate-induced acidification.
The addition of butyrate to the isolated parasites caused an extremely
rapid acidification (Fig. 5A);
pH
was attained within
4 s and was followed by a slow, progressive alkalinization,
reflecting the leakage of H+ out of the cell, down the
outward [H+] gradient. The pantothenate-induced
acidification was substantially slower than the butyrate-induced
acidification, taking ~150 s for
pH
to be reached in
the ATP-depleted parasites (Fig. 5A). Fig. 5B
compares the
pH
following the addition of increasing concentrations of pantothenate or
butyrate. In the case of pantothenate, the influx was sufficiently slow
for there to have been significant leakage of H+ out of the
parasite, down the [H+] gradient, during the ~150 s
required for
pH
to be
attained. To make a quantitative comparison of the relative magnitude
of the H+ uptake induced by the influx of butyrate and
pantothenate, it was therefore necessary to correct the
pH
measured following
addition of pantothenate, for the significant H+ leakage
that occurred during the period of pantothenate influx (as described in
the legend to Fig. 5). The corrected pantothenate-induced
pH
values (Fig.
5B, open circles) were very similar to the
butyrate-induced
pH
values (Fig. 5B, closed circles), consistent with
pantothenate and H+ entering the parasite with a
stoichiometry of 1:1. The transport process is therefore
electroneutral.

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Fig. 5.
Comparison of the effect of butyrate and
pantothenate on pHi of ATP-depleted, P. falciparum trophozoites suspended in a solution of pH
7.1. A, traces showing the
time-dependent decrease in pHi following the
addition of butyrate or pantothenate at the point indicated by the
closed arrowheads, each at a concentration of 40 mM. B, maximum decrease in pHi
( pH ) following the
addition of increasing concentrations of butyrate (closed
circles) or pantothenate (closed triangles). The
open circles show the
pH for pantothenate,
corrected for the alkalinization that occurred as a result of
H+ leakage out of the parasites during the extended time
period (~150 s) taken for the pantothenate-induced pHi to
reach its maximum value. In making the correction, the extent of
alkalinization to have occurred at each time point was estimated from
the (pH-dependent) alkalinization rates observed following
the fast acidification induced by the addition of different
concentrations of butyrate. This was then used to estimate the
pH that would have
been observed following the addition of pantothenate, had
H+ leakage not occurred. It is important to recognize that
pH is not a transport
rate. The non-linearity of the graph of
pH versus
[monocarboxylate] is not due to a saturation of the transport process
but is due, instead, to the fact that the buffering power of the
parasite cytosol ( i) increases in a non-linear manner
with decreasing pH (see "Experimental Procedures"). The data are
averaged from three separate experiments and are shown as mean ± S.E.
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The pantothenate-induced H+ influx was highly sensitive to
temperature. Upon reduction of the temperature from 37 °C to
5-10 °C, pantothenate-induced H+ influx was abolished
(Fig. 6A). An Arrhenius plot
was linear over the temperature range 16-37 °C (Fig. 6B)
and yielded an estimated activation energy for pantothenate-induced
H+ influx of 89 ± 6 kJ/mol (mean ± S.E.;
n = 3).

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Fig. 6.
Temperature dependence of
pantothenate-induced H+ influx into isolated P. falciparum trophozoites suspended in a solution of pH
7.1. A, traces, representative of those from
four separate experiments, showing the abolition of the
pantothenate-induced acidification on reduction of the temperature from
37 °C to 5 °C. B, Arrhenius plot for
pantothenate-induced acidification in the temperature range
16-37 °C. The pantothenate concentration was 40 mM. The
mean data (averaged from three separate experiments) are shown ± S.E. Linear regression of the data yielded an activation energy of
89 ± 6 kJ/mol.
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Phosphorylation of PA within the Parasite Is by a High Affinity
Kinase--
The kinetics of the phosphorylation of PA, within the
parasite, was investigated in parasite lysates. The rate of
phosphorylation was measured over the PA concentration range 0.1-0.75
µM, and the Michaelis-Menten equation was fitted to the
data (Fig. 7), yielding a
Km of 0.28 ± 0.03 µM and a
Vmax of 10.8 ± 0.7 µmol/(1012 parasites.h).

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Fig. 7.
Concentration dependence of the rate of
phosphorylation of [14C]PA by lysates of isolated
P. falciparum trophozoites. The data (averaged
from three separate experiments and shown as mean ± S.E.) were
fitted to the Michaelis-Menten equation, yielding a
Km of 0.28 ± 0.03 µM and a
Vmax of 10.8 ± 0.7 µmol/(1012 cells.h).
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DISCUSSION |
Pantothenic acid, one of the relatively few nutrients for which
the intracellular malaria parasite P. falciparum has an
absolute extracellular requirement (1), enters infected erythrocytes primarily via the new permeation pathways induced by the parasite in
the host cell membrane (7). From the host cell cytosol, PA is presumed
to enter the parasitophorous vacuole in which the parasite is enclosed,
via the large-diameter pores characterized previously in the vacuole
membrane (12). From the parasitophorous vacuole, PA is taken up across
the parasite plasma membrane. The results of this study shed light on
the mechanism by which this is achieved.
In this study we have used parasites functionally isolated from their
host cells by brief exposure of the parasitized cells to saponin.
Saponin permeabilizes the erythrocyte membrane to both low and high
molecular weight solutes (including host cell proteins), as well as
making the parasitophorous vacuole membrane (already highly permeable
to low molecular weight solutes (12)) permeable to proteins (8). By
contrast the parasite plasma membrane remains intact, able to support
transmembrane ion gradients (7). There is a possibility that the
solution within the parasitophorous vacuole compartment acts as an
unstirred layer, influencing the rate of passage of solutes into the
isolated parasites as well as the concentration of solutes in the
immediate vicinity of the parasite plasma membrane. However, the
finding in this study that butyrate equilibrates between the intra- and
extracellular solutions within 3-4 s, and the earlier observation that
NH3 does so even faster (in < 2 s (7)) would
indicate that our measurements of pantothenate transport (which is more
than an order of magnitude slower than that of either butyrate or
NH3) are not subject to significant unstirred layer effects
(13).
PA is transported across the plasma membrane of mammalian cells via a
well-characterized family of Na+:pantothenate symporters
((14-17) and see Fig. 1B). By contrast, the uptake of PA
into the intracellular malaria parasite is independent of
Na+, but is, instead, dependent on H+. The
demonstration, in Fig. 2A, that an inward [H+]
gradient can drive the active accumulation of [14C]PA (to
a distribution ratio
1) and the demonstration in Fig. 3 that an
inward [PA] gradient can drive the net influx of H+,
together, constitute good evidence for the operation of a
H+:pantothenate symport process, operating with a
stoichiometry of 1:1 (Fig. 5). These data do not distinguish between
simple diffusion of the undissociated acid, and a transporter-mediated process. However the findings that pantothenate-driven H+
influx is saturable (Km = 22.6 ± 2.3 mM; Fig. 3) and that both H+-driven PA influx
(Fig. 2A) and pantothenate-driven H+ influx
(Fig. 3A) are inhibited by phloretin and by sulfhydryl reagents are consistent with the involvement of a transporter. So too
is the finding that the transport process has a relatively high
activation energy (Ea = 89 ± 6 kJ/mol;
Fig. 6), which is in the range of those reported previously for
transporters in other systems and higher than that usually associated
with the process of simple diffusion (18).
The H+-coupling of pantothenate transport may allow the
parasite to take advantage of the H+ gradient normally
present across the parasite plasma membrane in accumulating PA from the
extracellular medium. The normal pHi of the parasite is
7.3-7.4 (Refs. 7, 19-21 and this study), compared with an estimated
pH of ~6.9 in a region of the erythrocyte cytosol immediately
adjacent to the parasite (21). There is therefore a significant inward
[H+] gradient, favoring the net accumulation of PA,
although the intracellular concentration of PA will be determined not
only by the stoichiometry of the transporter but by the relative rates of transport and metabolism of the vitamin under in vivo conditions.
The H+:pantothenate symporter of the intracellular malaria
parasite differs from the Na+:pantothenate symporters of
the host not only in its cation requirements, but in its substrate
affinity. Although the relatively high Km for the
process could not be measured with a high degree of accuracy (without
exposing the parasites to excessively high concentrations of the
pantothenate salt) it is evident that the transport of PA into the
parasite occurs via a low affinity process. The estimated Km of ~23 mM contrasts with those of
PA transporters elsewhere. The H+-coupled pantothenate
transporter of the yeast Saccharomyces cerevisiae has a
Km of 3.5 µM (22), whereas the
prokaryotes Lactobacillus plantarum and Escherichia
coli have Na+-coupled pantothenate transporters with
Km values of 0.8 µM (23) and 0.4 µM (24), respectively. The Na+-coupled
multivitamin transporters of mammalian cells have a
Km for pantothenate of 1.5-4.9 µM
(14-16), which falls within the normal range for the concentration of
PA in the blood plasma of healthy individuals (0.6-17 µM
(25)).
In contrast to the comparatively low affinity of the parasite's PA
transporter, the parasite's pantothenate kinase has a significantly higher affinity for pantothenate than its mammalian counterparts. The
kinase phosphorylates and thereby traps the PA within the parasite
cytosol, doing so with a Km (~0.28
µM) that is some 40-fold lower than that of the highest
affinity mammalian pantothenate kinase described to date (from rat
liver; Km ~12 µM (26)), as well as
being significantly lower than that of the high affinity transporters
of the cells of the host. Thus, although the transporter in the
parasite plasma membrane is a low affinity system, the combination of
the transporter and the kinase serves as a high affinity uptake system
that should enable the parasite to compete very effectively with the
cells of the host for this key nutrient.
The H+:pantothenate symporter described here is, to
our knowledge, the first example described of a H+-coupled
nutrient transporter in P. falciparum. The only previous description of a H+:pantothenate symporter in a eukaryote
cell is in the yeast S. cerevisiae (22). This is consistent
with the view that the physiology of the malaria parasite resembles
that of lower eukaryotes, such as plants and yeast (7), more closely
than it does that of mammalian cells. The PA transporters of mammalian
cells are Na+-dependent systems with
characteristics quite different from those of the
H+:pantothenate symporter described here. The finding here
that the pantothenate kinase of the parasite has a substrate affinity substantially higher than that of any of the mammalian pantothenate kinases described to date may be indicative of there also being significant differences between the mammalian and parasite forms of
this enzyme. Such differences, together with the previously demonstrated importance of PA for parasite growth (1), focus attention
on the possibility that the mechanisms involved in the uptake of PA by
the intracellular malaria parasite are suitable targets for new and
much-needed antimalarial drugs.