(Received for publication, November 7, 1994; and in revised form, December 16, 1994 )
From the
The proton extrusion mechanisms of Leishmania promastigotes were studied in terms of electrogenic movements of
protons and anions (Cl and
HCO
). Changes in membrane potential (V
) and intracellular pH
(pH
) were monitored fluorimetrically with the
potential sensitive dye bis-oxonol and the pH-sensitive dye
tetraacethoxymethyl 2`,7`-bis-(carboxyethyl)-5,6-carboxyfluorescein,
respectively. In nominal bicarbonate-free medium (pH
7.4, 28 °C), V
and pH
of Leishmania promastigotes were maintained at
-113 ± 4 mV and 6.75 ± 0.02, respectively. In
Cl
free (gluconate-based) medium, cells underwent a
time-dependent acidification (0.3 pH units) and a long term membrane
hyperpolarization (7-10 mV), both of which were greatly enhanced
in the presence of the anion blocker,
4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonic acid
(H
DIDS). Cells in Cl
-free medium
underwent a marked depolarization upon treatment with the
H
-ATPase inhibitor dicyclohexylcarbodiimide (DCCD),
but hyperpolarized after repletion with Cl
. In
Cl
-depleted cells, replenishment of Cl
led to a H
DIDS-sensitive cytoplasmic alkalinization
and a small initial hyperpolarization. Cells exposed either to DCCD or
to the H
uncoupler carbonylcyanide
chlorophenylhydrazone caused a marked cytoplasmic acidification and
membrane depolarization. In the presence of 25 mM HCO
, promastigotes maintained an
almost neutral cytosol, irrespective of H
pump action
or ionic composition of the medium. The present observations provide
evidence for the operation of a DCCD-sensitive electrogenic
H
-ATPase which contributes to the maintenance of a
highly hyperpolarized plasma membrane in Leishmania promastigotes. H
pump activity required a
parallel pathway of Cl
ions in order to dissipate the
pump generated electrical potential. In nominally CO
-free
media, the two electrogenic systems are implicated in the maintenance
of cell pH and indirectly in electrochemically driven nutrient uptake.
In physiological
CO
/HCO
-containing media, the
H
pump and Cl
channel play a role
only secondary to that of HCO
in
pH
homeostasis.
Leishmania parasites alternate between two major developmental stages, the flagellated promastigote which lives in the midgut of the sandfly vector and the obligatory intracellular nonmotile aflagellated amastigote which lives in the phagolysosome of mammalian macrophages. The successful adaptation and survival of the parasite in these distinctly different and extreme environments depends on the ability of these cells to acquire nutrients from the neighboring environment and in the maintenance of a constant internal milieu(1) .
In plants and lower eukaryotes, nutrients are
acquired by membrane transport mechanisms which involve coupling of
substrate with uptake of H down their electrochemical
gradient(2, 3) . The role of an electrogenic
H
-translocating ATPase in nutrient uptake has been
more conclusively demonstrated in bacteria(4) ,
fungi(5) , and plants(2, 6) . An analogous
H
-ATPase has been proposed in promastigotes of Leishmania(7, 8) , but direct experimental
evidence for H
pumping at the cell level has been
lacking. A role for a putative H
-pumping mechanism in
intracellular pH regulation has been previously hypothesized on the
basis of experiments carried out in nominally CO
-free
media(7, 8) , but the notion of an indirect
contribution of such a mechanism in H
-coupled nutrient
uptake has remained controversial(9, 10) . Moreover,
since Leishmania promastigotes naturally propagate in a
HCO
-rich environment, it is not clear to
what extent H
pumping subserves the maintenance of
cell pH.
Presently, little is known about the relative
permeabilities of the parasite plasma membrane to the prevailing ions
Na, K
, H
,
Ca
, HCO
, and
Cl
, let alone about passive or active ionic membrane
conductances. In this work we explored the electrogenic components
underlying H
-pumping mechanisms of intact Leishmania major promastigotes, that is H
pumping and anionic conductances. The studies were carried out
with intact cells placed in nominally CO
-free media of
different ionic composition and with the aid of the potentiometric
fluorescent dye, bis-oxonol(11) . Inhibitors of higher
eukaryotic H
pump and Cl
channels (12, 13) were used as tools for assessing the
respective contributions of both cationic and anionic transport systems
to V
and pH
. The
studies indicate that both, a DCCD(
)-sensitive H
pump and a 4,4`-diisothiocyanodihydrostilbene-2,2`-disulfonic
acid (H
DIDS)-sensitive Cl
channel
contribute electrogenically to acid secretion and to the maintenance of
a hyperpolarized plasma membrane and suggest a role for the two systems
in pH homeostasis and H
-coupled nutrient uptake in
nominally CO
-free media. In the more physiological
HCO
-containing media, while the
electrogenic role of the pump is maintained, pH regulation is primarily
supported by transporters of HCO
.
Figure 1:
Calibration of membrane potential V in Leishmania promastigotes.
An aliquot of 5
10
cells/ml was equilibrated with
0.1 µM bis-oxonol in NMG
solution. The
traces are of fluorescence intensity (540 excitation and 580 nm
emission) after background substraction following addition of
gramicidin (0.8 µg/ml) and KCl to give the final indicated
concentrations (inset). The main graph depicts the
relationship between fluorescence change (F) and the
calculated K
equilibrium potential V
, in millivolts (mV).
The ATPase inhibitor DCCD (10
µM), the protonophore CCCP (1 µM), and the
anion transport inhibitor HDIDS (0.5 mM) were
added directly to the medium containing cells and fluorescent dye prior
to measurements. None of these inhibitors or uncoupler interfered with
the fluorescence signal of bis-oxonol.
Figure 2:
Effect of cations on V. Cells were suspended in NMGCl medium,
pH 7.4, previously equilibrated with 0.1 µM bis-oxonol.
After signal stabilization was achieved, increasing concentrations of
either KGlu (potassium gluconate) or NGlu (sodium gluconate) were
added. V
was measured as described in the
legend of Fig. 1. Traces are representative of three separate
experiments.
Figure 3:
Effect of Cl depletion on V
and pH
. In parallel
experiments, cells were either preloaded with the pH indicator BCECF
and subsequently suspended in gluconate medium, pH 7.4 or were
suspended directly in gluconate medium, pH 7.4, containing bis-oxonol
(0.1 µg/ml). pH
and V
were continuously monitored as indicated under
``Experimental Procedures.''
Figure 4:
Effect of Cl replenishment on
the pH
and V
of
Cl
-depleted cells. Cells were preincubated in gluconate
medium, pH 7.4, for 30 min at 30 °C and a fraction loaded with
BCECF. Subsequently cells were resuspended in Cl
medium, pH 7.4, and pH
and V
were recorded as indicated in Fig. 3.
Figure 5:
Effect of HDIDS on
pH
and V
.
pH
and V
were
followed fluorimetrically in cells suspended in Cl
medium, pH 7.4, in the absence or presence of 0.5 mM H
DIDS.
Figure 6:
Effect of the H-ATPase
inhibitor DCCD and the protonophore CCCP on pH
and V
. Cells pretreated as described in Fig. 4were suspended in Cl
medium, pH 7.4,
either in the presence or absence of bis-oxonol. DCCD (10
µM) (A) or CCCP (1 µM) (B)
were added at the times indicated by the arrows, respectively.
Fluorescent changes were recorded as described in Fig. 3.
Direct evidence for the existence of a Cl conductive pathway was observed in cells in which the electrical
contribution of the pump was reduced by treatment with DCCD alone (Fig. 7A) or with both DCCD and CCCP (Fig. 7B). In Cl
-free medium, the
marked depolarization elicited either by a single agent or both agents
together, was followed by a significant hyperpolarization upon addition
of NMGCl (Fig. 7). Addition of an equivalent amount of sodium
gluconate produced only a minor change, due to a dilution effect. We
interpret the hyperpolarization to result from an inward negative
diffusion potential created by Cl
supplementation,
due to the existence of a Cl
conductive pathway in Leishmania promastigote plasma membranes. Essentially similar
results were obtained with cells suspended in isotonic mannitol-based
media (not shown).
Figure 7:
Hyperpolarization by addition of Cl to pump-inhibited cells. Cells suspended in gluconate medium, pH
7.4, containing bis-oxonol were treated with DCCD (10 µM),
either alone (A) or with CCCP (1 µM) (B). At the indicated time NMGCl (filled squares) or
NaGlu (open squares) were added (50 mM final). V
was determined fluorimetrically as
described in Fig. 3.
Figure 8:
Effect of HCO on pH
in Leishmania promastigotes. Upper, pH
was followed fluorimetrically
(as shown in Fig. 1) in Cl
medium (Cl
), pH 7.4, or gluconate-medium (Glu
), pH 7.4, either in the presence or absence
of 25 mM HCO
±
H
DIDS (0.5 mM). Lower, steady state
pH
values of cells incubated for 30 min in
Cl
medium at either pH 7.4 or 8.0
(pH
) in the presence (+) or absence(-)
of 25 mM HCO
.
In this work we studied the electrical properties of
H- and anion transport systems of Leishmania promastigotes. We based the studies on fluorescence measurements
of V
and pH
with the aid of
fluorescent potentiometric and pH metric dyes bis-oxonol (11) and BCECF(20) , respectively. We showed that both
H
and Cl
extrusion from cells are
electrogenic. A putative H
pump is shown to be
primarily responsible for generating a pronounced electronegative V
and a Cl
channel is shown to
subserve H
extrusion by dissipating the high potential
generated by the pump. Such coupling between H
and
Cl
fluxes has been demonstrated in a variety of cells
and in acidifying organelles of higher
eukaryotes(21, 22, 23) .
The highly
negative V of -113 ± 4 mV found in Leishmania promastigotes in steady state conditions agrees
with previous measurements of V
based on the
lipophilic cationic dye, TPP
in L. donovani promastigotes(18) . The maintenance of a
relatively high negative membrane potential seems to be a common
feature of several parasitic protozoa(24) , particularly in the
species related trypanosoma(25, 26) . In mammalian
cells, K
ion gradients and
K
-conductance, g
, are the major
determinants of the resting cell membrane potential(27) .
In Leishmania promastigotes, a substantial contribution of
g to V
could be implied on the basis
that the E
of -80 mV is close to V
. If that is the case, then a 10-fold change in
[K]
/[K]
should presumably
shift V
by about 60 mV. The fact that V
changed very little (this work) or only to a
minor extent (18 mV) in a previous study (18) would indicate
that g
contribution to V
is small
relative to other ionic conductances. Regarding g
, its
contribution to the resting V
is presumably also
small. This is shown in the minor hyperpolarization which follows when
cells are placed in Cl
-free media. We interpret this
to reflect g
limiting H
pumping (i.e. cell alkalinization) because of Cl
depletion
from cells. Thus, the role of g
is in supporting cation
(primarily H
) extrusion. This is best exemplified in
the acidification and membrane hyperpolarization which resulted from
placing cells in Cl
-free media or in
Cl
-containing medium in the presence of the anion
transport blocker H
DIDS ( Fig. 3and Fig. 5).
All this suggests that extrusion of H
s is probably the
major factor behind the highly negative V
generated across the Leishmania plasma membrane, while
the movement of anions would serve to partially dissipate this
potential and facilitate further H
extrusion. These
notions are consistent with and supported by the effects obtained with
DCCD and CCCP on both V
and pH
. In
this case, the partial abrogation of H
pump activity
by DCCD and the dissipation of H
gradients by CCCP led
to cell acidification and membrane depolarization. If the V
was partially clamped with CCCP, then addition
of extracellular Cl
to cells placed in gluconate
medium, should also lead to some hyperpolarization. This was indeed
observed (Fig. 7), indicating the presence of a
Cl
-conductive pathway.
Taken together, the
experimental evidence provided in this work supports the parallel
operation of an electrogenic H pump in conjunction
with a Cl
channel, whose combined action is acid
extrusion. As to the physiological role of such systems, they should be
evaluated in the context of the physiological media in which parasites
dwell(28, 29) . Parasites are major producers of acids (30) which would tend to acidify their cytosol. The present
work was carried out primarily in media not supplemented with
HCO
(although referred to as nominally
CO
-free, it had ambient dissolved CO
and
HCO
). Blocking of acid extrusion resulted
in cell acidification. However, when cells were suspended in
HCO
-supplemented media, pH
rose from 6.75 to about neutrality, and it was maintained in the
6.85-7.0 range even in the presence of an anion transport
inhibitor or when extracellular pH was raised to 8.0. These data
suggest that the physiological role attributed to H
pumping (7, 8, 14) and Cl
channels (14) in regulating the cytosolic pH of Leishmania promastigotes is substantial in relatively acidic
or HCO
-free media, but probably more
modest in HCO
-containing media than
previously hypothesized. The somewhat overestimated role of the systems
found in ambient (i.e. nominally CO
-free)
conditions is analogous to the role previously attributed to the
mammalian Na
-H
antiporter in pH
changes following signal transduction in the absence of
HCO
(31, 32) . Thus both
the H
pump and HCO
transporters are likely to play an important role in pH
homeostasis of Leishmania, by mediating acid neutralization
and/or extrusion. However, H
pumping by promastigotes
remained also fully active even in bicarbonate media, as judged by the
maintenance of a highly hyperpolarized membrane.
This
implies that the combined action of parasite H
pump
and Cl
channel might not only be important for pH
homeostasis per se. The marked membrane hyperpolarization and
possible generation of a proton motive force might subserve the active
uptake of substances, as previously
proposed(7, 8, 19) . Moreover, as these
transport systems subserve essential physiological functions, they
might be considered potential targets for pharmacological intervention.