(Received for publication, May 22, 1995)
From the
Following invasion by the malaria parasite there appear in the
parasitized erythrocyte new (``induced'') permeation pathways
that mediate the transport of a wide variety of small solutes. Although
anion-selective, these pathways have a significant cation permeability
and cause a substantial increase in the basal leak of cations into and
out of the infected cell. In this study of human erythrocytes infected in vitro with Plasmodium falciparum it was shown that
the transport of monovalent cations (Rb and choline),
but not that of a nonelectrolyte (sorbitol) or a monovalent anion
(lactate), via the malaria-induced pathways is strongly dependent on
the nature of the anion in the suspending medium. Substitution of
NO
for Cl
resulted in a
4-6-fold increase in the unidirectional influx and efflux of
Rb
, and a 2-3-fold increase in the influx of
choline via the induced pathways. By contrast, replacement of
Cl
with NO
caused a
slight (although not significant) decrease in the malaria-induced
influx of sorbitol and lactate. Hemolysis experiments with a range of
K
salts revealed that the net influx of K
into infected cells showed the same novel anion dependence as
seen for the unidirectional flux of Rb
and choline,
with hemolysis occurring much faster in iso-osmotic KNO
and
KSCN solutions than in KCl, KBr, or KI solutions. Hemolysis in the
corresponding Na
salt solutions was very much slower,
consistent with the induced pathways being selective for K
over Na
, and raising the possibility that the
efflux of cell K
via these pathways may play a role in
host cell volume regulation. A number of models that would account for
the anion dependence of malaria-induced cation transport are
considered.
Human erythrocytes infected with the mature (trophozoite) form of the malaria parasite, Plasmodium falciparum, show increased permeability to a diverse range of small solutes including polyols, amino acids, nucleosides, and monovalent anions and cations (reviewed by Ginsburg(1988, 1990, 1994), Cabantchik(1990), Gero and Upston (1992), Gero and Kirk(1994), and Elford et al.(1995)). The available data is consistent with the view that much of this increase is mediated by a single type of broad specificity permeation pathway. This pathway has not yet been identified at a molecular level but has the properties of an anion-selective pore or channel (Ginsburg et al., 1985) and shows functional characteristics similar in at least some respects to those of anion-selective channels in the plasma membranes of other cell types (Kirk et al., 1993, 1994).
Despite being strongly anion-selective, the malaria-induced pathway,
like a number of anion-selective channels elsewhere (Franciolini and
Petris, 1990), has a significant permeability to monovalent cations.
The membrane of the normal erythrocyte has a very low cation
permeability and the flux of cations via the induced pathway represents
a relatively large increase in the basal leak of cations into and out
of the infected cell. In this study we have investigated the
characteristics of malariainduced cation permeation in infected
erythrocytes. The induced transport of monovalent cations, unlike that
of the nonelectrolyte sorbitol and the monovalent anion lactate, was
found to vary dramatically with the nature of the permeant anion in the
suspending medium, increasing markedly when Cl was
replaced with either NO
or
SCN
. Mechanisms that might account for the unusual
ion permeability properties of the malaria-induced pathway are
considered.
Influx measurements were made under conditions designed to minimize
fluxes via the normal host erythrocyte transport systems. In Rb
influx experiments cells were
pretreated (for
10 min) with 0.1 mM ouabain to inhibit the
Na
/K
pump, 0.01-0.1 mM
bumetanide to inhibit the NaKCl
cotransporter, and
0.01-0.02 mM nitrendipine to inhibit the
Ca
-activated K
channel (Ellory et al., 1992). In choline influx experiments the use of an
extracellular choline concentration of 1 mM ensured that the
endogenous erythrocyte choline carrier (which has a K
of approximately 10 µM) was fully saturated and
therefore contributed little to choline uptake (Kirk et al.,
1994). Lactate influx into malaria-infected cells was measured in cells
pretreated (for 10 min) with 0.01-0.02 mM DIDS to
inhibit transport via the erythrocyte band 3 anion exchanger and 0.1
mM pCMBS to block the normal erythrocyte monocarboxylate
transporter (Poole and Halestrap, 1993).
Unidirectional influx rates
for all substrates were estimated from the amount of radiolabel
accumulated by the cells during a fixed incubation period, found in
preliminary experiments to fall within the initial linear phase of the
uptake time course. For Rb
and
[
C]choline the incubation period was 10 min, for
sorbitol, 1 min, and for lactate, 5-10 s. Influx experiments were
carried out at room temperature (
22 °C) unless specified
otherwise.
Radiolabeled solute influx was measured using two
different protocols. For Rb and choline, influx
experiments commenced with the addition of
Rb
or [
C]choline, together with unlabeled
substrate, to a microcentrifuge tube containing cells (±
furosemide). The final sample volume was 0.5 ml, the final cell
concentration typically 2
10
cells/ml, and the
final concentration of radioactivity was 1 µCi/ml. The flux was
terminated by transferring aliquots (0.11 ml) of the suspension to
microcentrifuge tubes containing 0.8 ml of ice-cold stopping solution
(HEPES-buffered, Cl
-containing saline supplemented
with 0.1 mM furosemide; Kirk et al.(1994)) layered
over 0.5 ml of dibutylphthalate. The tubes were centrifuged immediately
(10,000
g, 20 s) to sediment the cells below the oil.
For the more rapidly transported substrates, sorbitol and lactate
(Kanaani and Ginsburg, 1991), influx was measured using an alternative
approach. A microcentrifuge tube containing 0.15 ml of saline (with
radiolabeled substrate, unlabeled substrate and, where appropriate,
transport inhibitors), layered over 0.2 ml of dibutylphthalate, was
placed in a microcentrifuge. The flux commenced with the addition of an
aliquot of cell suspension, giving a final cell concentration of
approximately 2 10
cells/ml and a final
concentration of radioactivity of 1 µCi/ml. The flux was terminated
at the appropriate time by starting the centrifuge (10,000
g, 20 s), thereby sedimenting the cells below the oil. The
time taken between starting the centrifuge and termination of the flux
was estimated as 2 s (by extrapolation of data from short time course
experiments).
In all experiments, following sedimentation of the
cells below the oil, the aqueous supernatant solution was removed by
aspiration and the radioactivity remaining on the walls of the tube
removed by rinsing the tubes four times with water. The
dibutylphthalate was aspirated, then the cell pellet was lysed with
0.1% (v/v) Triton X-100 (0.5 ml) and deproteinized by the addition of
5% (w/v) trichloroacetic acid (0.5 ml), followed by centrifugation
(10,000 g, 10 min). Radioactivity was measured using a
-scintillation counter.
In the Rb
and [
C]choline influx experiments, the
extracellular space in the cell pellets was estimated from the amount
of
Rb
or
[
C]choline in pellets derived from aliquots of
the flux suspension sampled (into microcentrifuge tubes containing 0.8
ml of ice-cold stopping solution layered over 0.5 ml of
dibutylphthalate) and centrifuged within a few seconds of combining the
cells and radiolabel. In the shorter sorbitol and lactate influx
experiments, the amount of radiolabel trapped in the extracellular
space within the cell pellets was estimated from the amount of
[
C]sorbitol or [
C]lactate
in pellets sampled (within <2 s of combining the cells and the
radiolabeled substrate) from suspensions containing 0.1 mM furosemide to block influx (Kirk et al., 1994).
In order to average data obtained in experiments done on different parasitized cell cultures, the measured transport rates were corrected for parasitemia. The malaria-induced transport component was calculated (by subtracting the flux measured in uninfected cells from that measured in infected cell cultures) then divided by the fractional parasitemia to give the induced transport rate for the parasitized cells.
The rate constant for Rb
efflux, k
, was estimated from the slope of
the line fitted (by linear regression) to the graph of
ln([
Rb
]
(t)/[
Rb
]
(t = 0)) versus time, t, where [
Rb
]
(t = 0) denotes the total concentration of
Rb
present inside the cells at the
beginning of the efflux time course and [
Rb
]
(t) denotes the concentration of
Rb
present inside the cells at time t.
For
these experiments, iso-osmotic solutions of the Na and
K
salts of interest were prepared by dissolving the
salts to a concentration of 150 mM in a solution containing 10
mM HEPES and 5 mM glucose (pH 7.4). The pH was
readjusted to 7.4, then the osmolality adjusted (by the addition of
either the solid salt or the hypo-osmotic HEPES + glucose
solution) to lie within the range 298-306 mOsm
(kg
H
O)
.
Prior to beginning the time
courses, cells in RPMI (supplemented with 40 mM HEPES, 10
mM glucose, and 2 mM glutamine) were pretreated for
10-20 min at room temperature with ouabain (0.1 or 0.5
mM) and nitrendipine (0.02 mM) to inhibit the
erythrocyte Na/K
pump and
Ca
-activated K
channel, respectively
(Ellory et al., 1992). Time courses commenced with the
addition of a 0.2-ml aliquot of the infected cell suspension to 3.3 ml
of iso-osmotic Na
or K
solutions
(containing 0.1 mM ouabain and 0.01 mM nitrendipine)
to give a cell concentration of approximately 0.5
10
cells/ml. At predetermined intervals, 0.5-ml aliquots of the
suspension were transferred to microcentrifuge tubes containing 0.5 ml
of ice-cold stopping solution (400 mM sucrose in
H
O). The tubes were centrifuged for 30 s then 0.9 ml of the
supernatant solution was transferred to another tube for the subsequent
spectrophotometric (A
) estimation of hemoglobin
concentration.
In all such experiments the A value corresponding to full hemolysis of trophozoite-infected
erythrocytes was estimated from the final A
value achieved in the supernatant solution from infected cells
suspended in an iso-osmotic sorbitol solution (Ginsburg et
al., 1985; Kirk et al., 1994).
Human erythrocytes infected with P. falciparum trophozoites show increased rates of transport of a diverse range
of solutes. The available data is consistent with the view that much of
the increased transport is via a single type of pathway that is
inhibited by the anion transport blocker, furosemide (Kirk et
al., 1994). The phenomenon is illustrated in Fig. 1which
shows the rates of influx of four different solutes (two monovalent
cations (K(
Rb
) and
choline), a nonelectrolyte (sorbitol), and a monovalent anion
(lactate)) into uninfected cells and infected cells (±
furosemide) in the presence of reagents that inhibit the endogenous
transport systems of the normal human erythrocyte. As is clear from the
relative influx rates, induced transport is anion-selective: the
furosemide-sensitive flux of the monovalent anion, lactate, was several
orders of magnitude greater than that of the monovalent cations
K
(
Rb
) and choline, with
that of the uncharged polyol, sorbitol, falling in between.
Figure 1:
Rates of influx of the monovalent
cations K(
Rb
) and
choline, the non-electrolyte sorbitol and the monovalent anion,
lactate, into normal uninfected human erythrocytes (open bars)
and into P. falciparum-infected erythrocytes in the absence (closed bars) and presence (shaded bars) of 0.1
mM furosemide. The cells were suspended in a HEPES-buffered
saline (pH 7.4) containing 125 mM NaCl, 5 mM KCl, 5
mM glucose, and 25 mM HEPES.
K
(
Rb
) influx
measurements were carried out in the presence of ouabain (0.1
mM), bumetanide (0.01 mM), and nitrendipine (0.01
mM) to inhibit the endogenous erythrocyte
Na
/K
pump, NaKCl
cotransporter and Ca
-activated K
channel, respectively. Lactate influx measurements were carried
out in the presence of DIDS (0.02 mM) and pCMBS (0.1
mM) to inhibit the erythrocyte band 3 anion exchanger and
lactate transporter, respectively. The data are averaged from 6-9
experiments, each on cells from a different donor, and are shown
± S.E. The flux rates in each experiment were corrected to those
for a 100% parasitemia prior to averaging.
The
malaria-induced influx of the two cations, but not that of sorbitol or
lactate, increased markedly on replacement of Cl with
NO
in the suspending medium. This is
illustrated in Fig. 2which shows the furosemide-sensitive flux
component for the four solutes in the two different media. The influx
of K
(
Rb
) via the
furosemide-sensitive pathway was 4-6-fold higher in the
NO
medium than in the Cl
medium (p = 0.001, paired two-tail t-test). The induced influx of choline was also substantially
(2-3-fold) higher in NO
than in
Cl
(p = 0.005). By contrast, the
induced influx of both sorbitol and lactate was marginally (although
not significantly) slower in the NO
than
in the Cl
solution.
Figure 2:
Effect of replacing Cl with NO
on the furosemide-sensitive (i.e. malaria-induced) influx of
K
(
Rb
), choline,
sorbitol, and lactate into parasitized cells. Fluxes were measured in
malaria-infected erythrocytes washed (
4) then resuspended in
HEPES-buffered saline containing either Cl
(open
bars) or NO
(closed bars)
as the permeant anion. In each experiment the furosemide-sensitive
influx component was obtained by subtracting the flux measured in the
presence of 0.1 mM furosemide from that measured in its
absence and was corrected to 100% parasitemia. The data are averaged
from 6-7 experiments, each on erythrocytes from a different
donor, and are shown ± S.E. p values are those from a
paired (two-tail) t-test.
The data in Fig. 2are
from experiments carried out at 22 °C. In experiments at 37 °C
(not shown) a very similar (4-6-fold) stimulation of Rb
influx and (2-3-fold)
stimulation of choline influx was seen on replacement of Cl
with NO
.
One possible
explanation for the stimulatory effect of NO on the malaria-induced cation influx is that the replacement of
Cl
with NO
caused a
hyperpolarization of the cell membrane, resulting in an increased
electrical driving force for the influx of cations via a conductive
pathway. To investigate this possibility we tested the effect of
NO
on the efflux of
Rb
via the malaria-induced pathway. Fig. 3shows representative time courses for the efflux of
Rb
from cells preloaded with radiolabel,
washed in isotonic Cl
or NO
medium, then resuspended in the same solution with or without
furosemide. The inset shows the rate constants for the
furosemide-sensitive component of
Rb
efflux, averaged from four experiments on cells from different
donors. As in the influx experiments (Fig. 2), replacement of
Cl
with NO
caused a
4-6-fold increase in furosemide-sensitive
Rb
efflux (p < 0.001). The
stimulatory effect of NO
on cation influx
was therefore not due to a change in electrical driving force but
represents a genuine increase in the cation permeability of the induced
transport mechanism.
Figure 3:
Effect of replacing Cl with NO
on the efflux of
Rb
from malaria-infected erythrocytes.
The main figure shows representative time courses for the efflux of
Rb
from malaria-infected cells (82%
parasitemia) washed (
2) in solutions containing either
Cl
(open symbols) or
NO
(closed symbols), then
resuspended in the same solutions with (triangles) or without (circles) 0.1 mM furosemide. All solutions contained
0.1 mM bumetanide to inhibit the endogenous erythrocyte
NaKCl
cotransporter. The temperature was 37 °C. The inset shows averaged data for the furosemide-sensitive
component of
Rb
efflux from cells in a
Cl
(open bars) or NO
(closed bars) medium. The data are averaged from four
experiments, each on cells from a different donor, and are corrected to
100% parasitemia. The p value is from a paired (two-tail) t-test. Error bars denote
S.E.
The concentration dependence of the effect of
NO on the influx of
Rb
via the malaria-induced pathway is
shown in Fig. 4. Furosemide-sensitive
Rb
influx increased in a slightly curvilinear manner with increasing
NO
and decreasing Cl
concentration.
Figure 4:
Furosemide-sensitive
K(
Rb
) influx in cells
suspended in solutions containing varying concentrations of
Cl
and NO
. Cells were
washed four times in HEPES-buffered saline containing either
Cl
or NO
, then combined
in different proportions and incubated at approximately 25 °C for
15 min to allow equilibration of the permeant anions between the
intra- and extracellular solutions. Furosemide-sensitive influx was
obtained by subtracting the flux measured in the presence of 0.1 mM furosemide from that measured in its absence. Influx rates from
three different experiments, each on cells from different donors, were
corrected to 100% parasitemia before being averaged. Error bars denote S.E.
The effect of different anions on the induced cation permeability of malaria-infected erythrocytes was investigated in more detail using a hemolysis technique that has been used previously to investigate the induced permeability of malaria-infected erythrocytes (Ginsburg et al., 1985; Kirk et al., 1994). On suspension of cells in an iso-osmotic solution of a compound that is more permeant than the intracellular solutes, influx of solute into the cells exceeds efflux. This leads to uptake of water, cell swelling, and eventual lysis, the rate of which provides a semi-quantitative measure of the rate of influx of the extracellular solute(s).
Trophozoite-infected cells suspended in iso-osmotic
sorbitol solution at 22 °C hemolyzed within 30 min, consistent with
the high permeability of the malaria-induced pathway to this polyol (Fig. 5). Infected cells suspended in an iso-osmotic KNO solution (Fig. 5B) were almost fully hemolyzed
within 3 h but were protected against hemolysis by 0.1 mM furosemide, consistent with a role for the furosemide-sensitive
pathway in mediating the influx of KNO
. Cells in KSCN
hemolyzed at a similar rate to those in KNO
, while those in
KI, KBr, and KCl lysed more slowly. Uninfected cells were stable in all
of the media tested (not shown). The data indicate that the
malaria-induced transport of K
, like that of
Rb
, was strongly dependent on the nature of the
permeant anion present, being substantially greater in
NO
and SCN
than in
I
, Br
, or Cl
.
Figure 5:
Hemolysis of trophozoite-infected
erythrocytes in iso-osmotic solutions of different (A)
Na salts and (B) K
salts.
Cells in normal growth medium were suspended at t = 0
in iso-osmotic solutions of Cl
salt (
),
Br
salt (
), I
salt
(
), SCN
salt (
),
NO
salt (
),
NO
salt + 0.1 mM furosemide
(
), or sorbitol (
). All solutions contained ouabain (0.1
mM) and nitrendipine (0.02 mM) to block the
endogenous Na
/K
pump and
Ca
-activated K
channel,
respectively. Hemolysis is expressed as a percentage of that measured
in cells suspended in iso-osmotic sorbitol solution for 3 h. The data
shown are from a single experiment and are representative of those
obtained in three similar experiments on cells from different
donors.
Fig. 5A shows the results of a similar experiment
carried out with the corresponding Na salts. For
infected cells suspended in NaNO
or NaSCN there was slight
hemolysis after 3 h. Cells in iso-osmotic solutions of the other
Na
salts, or in an iso-osmotic NaNO
solution containing 0.1 mM furosemide, remained stable
throughout the 3-h incubation.
The membrane of the normal (uninfected) human erythrocyte has a relatively low permeability to cations. Thus, although the pathway that mediates the increased permeability of P. falciparum-infected erythrocytes shows a marked preference for anions and nonelectrolytes over cations (Ginsburg et al., 1985; Kirk et al., 1994; Fig. 1and Fig. 2of the present study), the low but significant flux of cations via this pathway represents a large relative increase in the basal cation permeability of the infected cell.
In this study the rate of
transport of monovalent cations in malaria-infected cells was found to
be strongly dependent upon the nature of the anion present. The
unidirectional influx and efflux of Rb
via the induced pathway increased 4-6-fold on replacement
of Cl
with NO
in the
medium (Fig. 2Fig. 3Fig. 4). The same maneuver
caused a 2-3-fold increase in induced choline influx (Fig. 2). The observation that both the influx and efflux of
Rb
were affected to the same extent
indicates that the effect cannot be explained in terms of altered
electrical driving forces following anion substitution, but represents
a genuine difference in the cation permeability of the pathway in the
presence of the different anions.
The time courses of hemolysis of
parasitized cells in iso-osmotic solutions of a range of different
K salts (Fig. 5B) indicate that the
net flux of K
via the malaria-induced pathway showed a
similar anion dependence to that seen in the (unidirectional)
Rb
flux experiments. They also provide
further information on the effects of different anions on the pathway:
induced K
transport was substantially greater in
NO
and SCN
than in
I
, Br
, or Cl
media.
The anion dependence of K,
Rb
, and choline transport via the malaria-induced
pathway is quite unlike that of the well known cation:anion
cotransporters which, in contrast to the induced pathway, carry cations
much better in the presence of Cl
(and
Br
) than in the presence of
NO
or SCN
.
In marked
contrast to the situation in the various iso-osmotic K salt solutions, trophozoite-infected cells in iso-osmotic
solutions of the corresponding Na
salts showed little
if any hemolysis over a 3-h period (Fig. 5A). Without
knowing the respective fates of Na
and K
ions once inside the infected erythrocyte, it is not possible to
draw quantitative conclusions about the rates of transport of these two
cations. However, the data are consistent with the malaria-induced
pathway having a substantially higher permeability to K
than to Na
. It is unclear how a pathway that is
permeable to molecules as large as nucleosides (Kirk et al.,
1994; Upston and Gero, 1995) might discriminate between these two
alkali metal cations. However, whatever the physical basis of this
apparent selectivity, it may have important implications for the
physiological role of the induced pathway. Any ``leak''
pathway that is present in the cell membrane and that is selective for
K
over Na
has the potential to play a
role in cell volume regulation. Under physiological conditions such a
pathway would mediate the net efflux of K
(with
Cl
), down its electrochemical gradient (while not
allowing a corresponding influx of Na
), resulting in a
loss of cell water and a consequent decrease in cell volume. The
present results therefore raise the possibility that the induced
transport pathway plays an important role in host cell volume
regulation, mediating the net efflux of K
from the
parasitized cell (as well as that of amino acids resulting from the
digestion of hemoglobin; Zarchin et al.(1986)), thereby
countering the effect on cell volume of the growth of the intracellular
parasite.
In contrast to the dramatic increase in the rate of
induced cation transport on replacement of Cl with
NO
(or SCN
), the
induced transport of the nonelectrolyte, sorbitol, and the monovalent
anion, lactate, was slightly (although not significantly) slower in
NO
than in Cl
media (Fig. 2). Any model of the malaria-induced pathway should
therefore account for why the permeation of cations is strongly
anion-dependent, whereas the permeation of nonelectrolytes and anions
is not. Fig. 6A shows a schematic representation of the
induced pathway in which it is represented as an anion-selective
channel bearing a positive charge or dipole, as well as a hydrophobic
region, represented by the shaded area. The positive charge or
dipole would provide the anion-selectivity filter, allowing the passage
of anions and uncharged solutes, but repulsing cations. The hydrophobic
region would account for the previously described preference of the
pathway for hydrophobic over similarly sized hydrophilic solutes
(Ginsburg et al., 1985; Ginsburg and Stein, 1987; Kirk et
al., 1994).
Figure 6: Alternative models of solute permeation through the malaria-induced pathway. A, the pathway is represented as a channel containing a hydrophobic region (shaded area) and a positive charge or dipole which provides the anion selectivity filter, allowing the passage of anions and uncharged solutes, but repulsing cations. In the model represented by B, (i), permeant anions enter the channel and bind at the positive site, thereby neutralizing it and allowing the flux of cations through the pathway. In the model represented by B, (ii), cations permeate the channel as electroneutral cation:anion pairs. Both models would account for the pronounced anion dependence of cation permeation of the induced pathway, as well as the observation that the transport of nonelectrolytes and anions is little affected by anion substitution. C, shows the interaction of an inhibitor with the channel. The most potent inhibitors so far identifed of malaria-induced solute transport all have a negative charge at one end and a relatively large hydrophobic tail (shaded). The negative group would be expected to interact with the cationic site on the channel and the hydrophobic tail with the hydrophobic region of the pathway.
There are a number of ways in which anions might facilitate the passage of cations via a channel of this sort, by interacting either with the channel or with the permeating cation. As represented in Fig. 6, B (i), a permeant anion may enter the channel and bind at the cationic site, thereby shielding the positive charge and thus allowing the passage of cations through the pore. Alternatively, as represented in Fig. 6, B (ii), a monovalent cation might interact with a permeant anion to form a transient, neutral cation:anion pair which is able to permeate the channel.
In both models the rate of cation transport
through the pathway might be expected to depend upon the nature of the
anion present. In model (i) the cation permeability would depend upon
both the ability of the anion to enter the channel and the strength and
duration of its interaction with the cationic site. This model would
account for the cation transport results obtained in the present study
if it were postulated that NO and
SCN
interact more strongly with the cationic site
than Cl
, Br
, or
I
. In model (ii) the cation permeability would depend
upon the propensity of the permeant anion to form an ion pair with the
cation (either in solution or perhaps within the microenvironment of
the channel itself), and upon the permeability of the pathway to this
transient complex. This model would account for the cation transport
results obtained here if it were postulated that cation:anion pairs
involving NO
or SCN
form more readily and/or permeate the channel more easily than
those with Cl
, Br
, or
I
.
Both models would account for the lack of
stimulation of the flux of nonelectrolytes and anions via the pathway
under conditions in which cation transport is increased. If, in model
(i), the permeant anion interacting with the cationic site actually
restricts the passage of solutes through the pathway, substitution of
Cl with an anion that spends a greater time at this
site might slow the flux of non-cationic solutes through the pathway
(as was observed for sorbitol and lactate on substitution of
Cl
with NO
; Fig. 2). In model (ii) the nature of the anion is unlikely to
affect the permeation of nonelectrolytes and anions, unless the
cation:anion pairs have some tendency to block the pathway.
Fig. 6C shows a representation of the interaction of
an inhibitor with the channel. The most potent inhibitors so far
identified of malaria-induced solute transport (including furosemide)
are all monovalent anions with a carboxylate group at one end and a
relatively large hydrophobic ``tail.'' The anionic
carboxylate group would be expected to interact with the cationic site
on the channel and the hydrophobic tail with the hydrophobic region of
the pathway. A recent structure-activity analysis with a series of
analogues of the widely used Cl channel blocker,
5-nitro-2-(3-phenylpropylamino)benzoic acid, indicated that the potency
with which these compounds inhibited the malaria-induced pathway
increased with the length and hydrophobicity of the tail (Kirk and
Horner, 1995). This is consistent with the present model in which
increasing the hydrophobicity of the inhibitor might be expected to
strengthen the hydrophobic interaction between the inhibitor and the
channel.
The basic features of the model represented in Fig. 6are not fundamentally different from those of the model proposed by Ginsburg and Stein(1987), in which the malaria-induced transport of solutes was postulated to occur via the protein-lipid interface of parasite-derived proteins inserted into the host erythrocyte membrane. The hydrophobic region shown in Fig. 6could, as in the Ginsburg-Stein model, be provided by the lipid bilayer, rather than by a protein component as would be assumed to be the case in a conventional model of an ion channel. Given the marked similarities between the substrate selectivity and pharmacological properties of the malaria-induced pathway and those of a number of anion-selective channels elsewhere (e.g. Kirk et al. (1992b)), it is perhaps worth considering whether a similar model (in which solutes traverse the membrane via a pathway formed at a protein-lipid interface) might be applicable to some of these other channels.
In summary, the transport of cations but not
that of nonelectrolytes or anions via the pathway induced in human
erythrocytes by the malaria parasite showed a marked dependence on the
nature of the anion in the suspending medium, increasing severalfold
when Cl was replaced by NO
or SCN
. A number of models of ion permeation
might account for this effect. A comparison of the rates of hemolysis
of malaria-infected cells suspended in iso-osmotic solutions of a range
of Na
and K
salts indicates that the
new permeation pathway is selective for K
over
Na
. This is consistent with a physiological role for
this pathway in mediating the volume regulatory efflux of K
from the malaria-infected cell and thereby playing an important
role in host cell volume control.