transport of diverse substrates into malaria-infected erythrocytes

Tm JOWAL OF BIOUXICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 5 , Issue of February 4, pp. 3339-3347, 1994 Printed in U S A .

Transport of Diverse Substrates into Malaria-infected Erythrocytes via a Pathway Showing Functional Characteristics of a Chloride Channel*

(Received for publication, August 6, 1993)

Kiaran Kirk$$, Heather k Homer$, Barry C. Elfordnll, J. Clive Ellory$, and Chris I. Newboldn From the $University Laboratory of Physiology, Oxford, OX1 3PI: United Kingdom and Vnstitute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 SOU, United Kingdom

Following infection by the malaria parasite, Plasnw- dium falciparum, human erythrocytes show increased permeability to a variety of low molecular weight solutes. In this study a number of anion transport blockers were identified as potent inhibitors of the transport of a wide range of solutes into human erythrocytes infected in vitro with €? falciparum. S-Nitro-2-(3-phenyl- propy1amino)benzoic acid (NPPB), furosemide, and niflumate blocked the malaria-induced transport of monovalent cations, neutral amino acids, sugars, nucleosides, and monovalent anions. For all of the substrates tested the order of potency of these three inhibitors was the same (NPPB > furosemide > niflumate) and dose-response curves for the effect of these inhibitors on malaria-induced choline transport were similar to those for malaria-induced thymidine transport. The data suggest that much, if not all, of the high capacity (non-saturable) transport of low molecular weight solutes into l? falciparum-infected erythrocytes is via a single type of pathway. The broad specificity of the pathway, its non-saturability in the physiological concentration range, and its failure to distinguish between stereoisomers (L- and D-alanine) are consistent with its being a type of pore or channel. For those substrates for which quantitative influx measurements were made the magnitude of the malaria-induced (inhibitor-sensitive) transport was in the order: C1- > lactate > thymidine, adenosine > carnitine > choline > K'. The pathway is therefore anion-selective. The pharmacological and substrate-selectivity properties of the pathway show marked similarities to those of chloride channels in other cell types; this raises the possibility that the high capacity transport of small organic solutes may be an important and, as yet, largely unrecognized role for such channels in other tissues.

During the intra-erythrocytic development of the malaria parasite, Plasmodium falciparum, the red cell shows increased permeability to a wide range of structurally unrelated solutes via transport pathways that are functionally distinct from those known to operate in normal erythrocytes. Solutes which

92/2/15 and 036615/W92/2/1.5 and by The University of Oxford. The * This work was supported by The Wellcome Trust Grants 036078W

costs of publication of this article were defrayed in part by the payment

tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate of page charges. This article must therefore be hereby marked "udver-

this fact. $3 Lister Institute Research Fellow and Staines Medical Research Fel-

low (Exeter College, Oxford). To whom correspondence should be ad- dressed: University Laboratory of Physiology, Parks Rd., Oxford, OX1 3PT, UK. Tel.: 44-865-272438; Fax: 44-865-272469.

11 Supported by The Medical Research Council.

have been shown to enter infected cells via the new permeation pathways include sugars and sugar-alcohols (Ginsburg et al . , 1983, 1985; Ginsburg, 1988), amino acids (Elford et al., 1985; Ginsburg et al., 1985), nucleosides (Gero et al., 1988, 1991; Gero, 1991; Gero and Upston, 19921, anions (Kutner et al., 1983; Ginsburg and Stein, 1987a; Cabantchik, 1990; Kanaani and Ginsburg, 1991; Elford and Pinches, 1992; Poole and Halestrap, 19931, and cations (Bookchin et al. 1980; Elford et al . , 1990a, 1990b; Kirk et al . , 1991a, 1991b, 1992a). I t has been suggested that the new pathways facilitate both the entry of metabolic and biosynthetic substrates into infected cells and the rapid exit of potentially harmful catabolites (Elford et al., 1985; Ginsburg et a l . , 1985; Ginsburg and Stein, 1987b; Sher- man, 1988; Ginsburg, 1988; Cabantchik, 1990; Kanaani and Ginsburg, 1991; Poole and Halestrap, 1993). However, despite their possible physiological importance and chemotherapeutic potential (Cabantchik, 1989), neither the identity nor the bio- physical nature of the parasite-induced pathways has been conclusively established.

In early studies of the enhanced permeability of human erythrocytes infected in vitro with P. falciparum it was suggested that the enhanced transport of monosaccharides and neutral amino acids might occur via simple positively charged pores which exclude cations (Kutner et al . , 1983; Ginsburg et al . , 1985). However, this model was rejected later in favor of one in which the transport of small organic solutes occurs via pathways formed from a mismatch between the lipids of the erythrocyte membrane and parasite-derived integral membrane pro- teins (Ginsburg and Stein, 1987b; Ginsburg, 1990). More recently it has been proposed that a combination of these mechanisms might operate, with hydrophilic substrates cross- ing the infected erythrocyte membrane via pores, and hydrophobic compounds permeating via protein-lipid interfaces (Ca- bantchik, 1990; Cabantchik et ul., 1990). However, alternative explanations have also been postulated, ranging from pertur- bation of the arrangement of phospholipids in the host cell membrane (Taraschi et al., 1986; Joshi et al . , 1987) to insertion (or activation) of heterogeneous, substrate-specific transporters (Elford et al., 1991; Poole and Halestrap, 1993).

In this work we address the question of whether the malaria- induced, high capacity (non-saturable) transport of low molecular weight solutes into parasitized erythrocytes occurs via com- mon routes or via heterogeneous, functionally distinct transport pathways. We have identified a number of potent inhibitors of the enhanced transport in malaria-infected erythrocytes (including the most effective inhibitor reported to date) and have shown that these compounds block the movement of a diverse range of solutes into infected cells. Our data are consistent with the hypothesis that much of the malaria-induced transport of cations, anions, amino acids, sugars, and nucleosides into parasitized cells occurs through a single type of pathway. This pathway behaves as an anion-selective chan-

3339

3340 Induced Dansport in Malaria-infected Erythrocytes

ne1 and shows marked functional similarities to chloride channels in other cell types.

A summary of parts of this work has been published previously in abstract form (Kirk et al., 199213).

EXPERIMENTAL PROCEDURES Materials-86RbC1 was from DuPont NEN. [14C]Choline chloride,

~-[“C]carnitine, [l4C1adenosine, [3H]thymidine, sodium ~-[l~C]lactate, and NaW1 were from Amersham Corp. Ouabain, 4,4’-diisothiocyanatostilbene-2,2’-disulfonic acid (DIDS),’ and 4-acetamido-4’-isothiocyanatostilbene-2,2’-disulfonic acid (SITS) were from Sigma. Bu- metanide was a giR from Leo Laboratories (Aylesbury, Buckinghamshire, UK). Nitrobenzylthioinosine (NBMPR) was a giR from Dr J. D. Young (Department of Physiology, University of Alberta, Canada). 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) was a giR from Prof. R. Greger (Physiologisches Institut der Albert-Ludwigs- Universitat, Freiburg, Germany) and indacrinone (MK-196) was a & from the late Dr. M. W. Wolowyk (Faculty of Pharmacy and Pharma- ceutical Science, University of Alberta, Canada).

Cell Culture-Human erythrocytes (type 0) infected with the IT04 line of I? falciparum (Berendt et al., 1989) were cultured under 1% Oz, 3% COz, 96% Nz in RPMI 1640 culture medium (Life Technologies, Inc.) supplemented with HEPES (40 man), glucose (10 man), glutamine (2 man), gentamicin sulfate (25 mditer), and serum (10% v/v, pooled from different blood donors, Blood Transfusion Service, Oxford and Bristol). All experiments were carried out using synchronized suspensions of trophozoite-infected cells (approximately 35 h post-invasion; 38-95% parasitemia), prepared using a combination of sorbitol hemolysis (Lambros and Vanderberg, 1979) and gelatin flotation (Pasvol et al., 1978) as described elsewhere (Kirk et al., 1992a).

In experiments comparing uninfected cells with malaria-infected ma- terial, erythrocytes (from the same donor) were incubated in parallel with the infected red cell cultures under identical conditions for at least 24 h prior to the experiment. Cell concentrations were determined using either a Coulter counter (model Zm) or an improved Neubauer counting chamber. Parasitemia was estimated from methanol-fixed Giemsa- stained smears.

Radioisotope Influx MeasurementeThe unidirectional influx into infected and uninfected erythrocytes of the monovalent cations K+ and choline, the zwitterion L-carnitine, the uncharged nucleosides adenosine and thymidine and the monovalent anions L-lactate and C1- was estimated from the uptake of =Rb+, [14Clcholine, ~-[‘~C]carnitine, [14Cladenosine, [3Hlthymidine, ~-[‘~Cllactate and 36Cl-, respectively, into cells washed four times by centrifugation (5 min at 1000 x g ) then resuspended in HEPES-buffered saline (125 man NaCl, 5 m~ KC1,25 man HEPES, 5 man glucose; pH 7.4).

Influx time courses were obtained using one of two methods. In the first, a series of 6 1 0 microcentrifuge tubes was prepared, containing 0.15 ml of saline (with radiolabel, unlabeled substrate and, where appropriate, transport inhibitors), layered over 0.2 ml of dibutylphthalate. The samples were pre-equilibrated to the required temperature then, at predetermined intervals, an aliquot of cell suspension (0.15 ml, equilibrated to the same temperature) was dispensed into each tube in turn. Immediately following the addition of cells to the final tube in the series the samples were centrifuged (10,000 x g , 30 s) to sediment the cells below the oil and thereby terminate the flux. 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 the second time course method cells in HEPES-buffered saline were combined with the appropriate reagents in a single microcentrifuge tube at 37 “C and allowed at least 10 min to equilibrate to temperature. The flux commenced with the addition of radiolabeled substrate. The sample was mixed thoroughly then, at regular intervals, aliquots of the suspension were transferred to microcentrifuge tubes containing 0.8 ml of ice-cold “stopping solution” (isotonic saline supplemented with 100 p~ furosemide) layered over 0.25 ml of dibutylphthalate and these were centrifuged immediately (10,000 x g , 30 8) .

In both methods, 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 was removed by rinsing the tubes four times with water. The dibutylphthalate was aspirated,

2,2’-disulfonic acid; MK-196, indacrinone; NBMPR, 6[(4-nitroben- The abbreviations used are: DIDS, 4,4’-diisothiocyanatostilbene-

zyl)thio]-9-~-~-ribofuranosylpurine (nitrobenzylthioinosine); NPPB,

isothiocyanatostilbene-2,2‘-disulfonic acid. 5-nitro-2-(3-phenylpropylamino)benzoic acid; SITS, 4-acetamido-4‘-

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 xg , 10 min). Radioactivity was measured using a p-scintillation counter.

In some experiments initial unidirectional influx rates were estimated from the amount of radiolabel accumulated during a fixed incubation period that fell within the initial (approximately linear) portion of the influx time course. The incubation periods used were normally 3-5 min for choline influx at 37 “C (see Fig. IA), 1 0 3 0 min for choline influx at 22 “C, 8-12 min for Rb+ influx at 37 “C (see Fig. lB), and 4-5 s for thymidine influx at 22 “C (see Fig. 6C). Fixed-period incubation experiments with the rapidly transported substrate thymidine were carried out by adding 0.15 ml of cell suspension to 0.15 ml of saline (containing radiolabel, unlabeled substrate, and inhibitors as appropriate) layered over 0.2 ml of dibutylphthalate in a microcentrifuge tube. 4-5 s later the tube was centrifuged (10,000 x g, 30 s) to sediment the cells below the oil and thereby terminate the flux. Fixed period choline and Rb’ influx experiments commenced with the addition of radiolabel together with unlabeled substrate to a microcentrifuge tube containing cells together with the appropriate pharmacological reagents. The final sample volume was 0.5 ml. Influx was terminated at the appropriate time by transferring aliquots (0.11 ml) of the suspension to microcentrifuge tubes containing 0.8 ml of ice-cold stopping solution layered over 0.25 ml of dibutylphthalate. The tubes were centrifuged immediately then processed for scintillation counting as outlined above.

In all influx experiments the amount of radiolabel trapped in the extracellular space within the cell pellets was estimated from “zero time” samples which contained the transport inhibitors identified in this study (at maximally effective concentrations), with sampling within a few seconds of combining the cells and the radiolabeled substrate.

Iso-osmotic Hemolysis Measurements-A simple, semi-quantitative hemolysis method which has been used extensively in previous studies of the induced permeability of malaria-infected erythrocytes (Ginsburg et al., 1985) was used to monitor the transport of choline, carnitine, various amino acids (L-glutamine, L-serine, L-threonine, L-alanine, D- alanine, L-valine, and L-methionine), sugars and sugar alcohols (sucrose, D-galactose, D-glucose, msorbitol, and o-ribose), and nucleosides (thymidine and uridine) into infected cells. Iso-osmotic substrate solutions were prepared by dissolving the compounds to a concentration of either 160 man (in the case of choline chloride) or 300 man (in the case of all other solutes) in HzO containing 10-20 man HEPES (pH 7.41, then adjusting the osmolality (by the addition of either the solute or the HEPES solution) to 310 10 mOsm (kg HzO)”, measured using a Wescor vapor pressure osmometer (5100 C). Hemolysis time courses commenced with the addition of a small volume of malaria-infected cells (suspended at a hematocrit of around 10% in normal growth medium) to the iso-osmotic solutions, at 37 “C. The final hematocrit was around 0.5%. The iso-osmotic thymidine and uridine solutions contained NBMPR (20 p ~ ) , a potent inhibitor of the endogenous erythrocyte nucleoside transporter (Jarvis et al., 1982; Gero et al., 1988), and the iso-osmotic glucose, galactose, and ribose solutions contained cytochalasin B (20 p ~ ) , a potent inhibitor of the endogenous erythrocyte sugar transporter. Aliquots of the suspension were sampled at regular intervals into microcentrifuge tubes which were then centrifuged (10,000 x g, 30 8). The supernatant solution was removed, and the hemoglobin in this solution, and hence the degree of hemolysis, estimated from the absorbance at 540 nm. Normal, uninfected erythrocytes incubated under the same conditions remained stable (i.e. did not lyse) for at least 1 h (results not shown). Hemolysis of the infected cells may therefore be attributed to the net influx of substrates via pathways induced by the parasite.

RESULTS

Malaria-induced Cation Dunsport-Fig. 1 shows time courses for the transport of monovalent cations, choline and K+[86Rb+], into normal erythrocytes and into cells infected with €? falciparum. When choline was presented at an extracellular concentration of 1 IMI (sufficient to saturate the native erythrocyte choline transporter; Kirk et al. (1991b), its rate of uptake into infected cells was manyfold higher than that into uninfected cells (Fig. lA). In similar experiments with red cells from 19 different donors the unidirectional influx of choline, measured at 37 “C in synchronized trophozoite-infected cell suspensions at 5695% parasitemia ranged from 60 to 218 pmol/(1012 RBC.h), with a mean value (&E.; n = 19) of 145 f 10 pmov

Induced llansport in Malaria-infected Erythrocytes 3341

(A) choline

0 2 4 6 8 1 0

Time ( m i n )

40

30

20

10

0

(B) K+("Rb+)

0 10 20 30 40

Time (min ) FIG. 1. A, time courses for the influx of choline into uninfected eryth-

rocytes (V) and malaria-infected erythrocytes (40% parasitemia, 0) at 37 "C. The extracellular choline concentration was 1 m. B , Time courses for the influx of K+[=Rb+] into uninfected erythrocytes (V) and malaria-infected erythrocytes (63% parasitemia, 0) at 37 "C. The extracellular K+ concentration was 5 m ~ . The samples contained ouabain (100 p ~ ) , bumetanide (100 p ~ ) , and nitrendipine (10 p x ) to block the flux of =Rb+ via the Na+/K+ pump, the NaKCl, cotransporter, and the Ca2+-activated K+ channel, respectively.

RBC.h). In uninfected cells from the same donors the unidirectional choline influx measured under the same conditions ranged from 1.3 to 7.4 pn0l/(10'~ RBC-h), with a mean value of 3.8 k 0.4 pmol/(1012 RBC-h).

A similar increase was seen in the basal permeability of the infected cell to 86Rb+ (which provides a measure of K+ permeability). Fig. 1B shows time courses for the influx of K+PRb+] into infected and uninfected cells pretreated with ouabain, bumetanide, and nitrendipine in order to inhibit the native erythrocyte Na+/K+ pump, NaKC12 cotransporter, and Ca2+-activated K+ channel, respectively (Ellory et al., 1992). In experiments with blood from 11 different donors, the unidirectional influx of K+ measured at 37 "C in trophozoite-infected cell suspensions (70-95% parasitemia) in the presence of 100 p ouabain, 100 p bumetanide, and 5 m~ K+, ranged from 119 to 223 pn0l / (10~~ RBC.h), with a mean value of 175 10 pnoV

RBC.h). Under the same conditions, the influx of K+ into uninfected cells from the same donors ranged from 12 to 29 pmoV(1012 RBC.h), with a mean value of 17 2 2 pmol/(1012 RBC.h).

Various membrane transport inhibitors were screened for their effect on the increased cation permeability of malaria- infected cells, and a number of potent inhibitors were identified. Somewhat surprisingly, these were all compounds familiar as anion transport inhibitors. The effect of selected anion transport inhibitors on the malaria-induced components of choline and K+Ls6Rb+1 transport into infected erythrocytes is shown in Table I. DIDS (at a concentration far in excess of that usually necessary to block anion exchange via the band 3 protein; e.g. Schofield et al. (1992)) caused partial inhibition of malaria- induced choline and s6Rb+ influx. Bumetanide, MK-196, niflumate, furosemide, and NPPB were more effective inhibitors of the induced choline flux, and the latter four reagents also blocked most of the induced transport of 86Rb+.2 Dose-response curves for the effect of niflumate, furosemide, and NPPB on malaria-induced choline influx are shown in Fig. 2. The IC5o values for the three reagents (i.e. the concentrations required to reduce the induced choline influx by 50%) were for niflumate, 20 p; for furosemide, 5 p; and for NPPB, 0.8 p.

Malaria-induced Zwitterion IFanspoTt-Having identified a number of potent inhibitors of parasite-induced cation trans-

' The =Rb+ influx experiments were all camed out in the presence of 0.1 m bumetanide in order to block the erythrocyte NaKC1' cotransporter. The effect of an additional 0.1 m bumetanide was not tested.

TABLE I Effect of anion transport inhibitors (100 p ~ ) on the malaria-induced

influx of choline and K+P6Rb+l at 37 "C The induced flux was calculated by subtracting the influx rate meas-

ured in uninfeded erythrocytes (cultured in parallel with infected cells) from that in infected cells from the same donor. Induced influx rates (mean values from n experiments, each on cells from different donors, mean f S.E.) are expressed as a percentage of those measured in the absence of inhibitor. Choline fluxes were measured at an extracellular choline concentration of 1 m ~ , under which conditions the influx of choline into infected cells was generally > 20-fold higher than that into uninfected cells. K+[=Rb+] fluxes were measured in the presence of ouabain (100 p ~ ) and bumetanide (100 p ~ ) to inhibit the endogenous Na+/K+ pump and the NaKCl, cotransporter, respectively; under these conditions the influx of Rb+ into infected cells was typically 10-fold higher than that into uninfected cells.

Inhibitor Induced choline influx Induced K+PRb+] influx

Z control DIDS 54 f 3*= ( n = 3) Bumetanide 45 f 2*** ( n = 6)

5 7 * 4 ( n = 3 )

Niflumate 16 2 2*** ( n = 4) Furosemide 7 * 1*** ( n = 7)

31 f 9* ( n = 5) 22 e 4*** ( n = 9)

NF'PB 7 f 2** ( n = 4) 17 f 4* ( n = 4)

- MK-196 35 2 4*** ( n = 3) 49 f 10* ( n = 3)

a In all cases the induced flux measured in the presence of each of the transport inhibitors was significantly less than that measured in their absence (paired t test; *, p < 0.05; **, p < 0.01, ***, p < 0.001).

100 4 L c .-

h

0 0 u c a)> 3 0 - 0 0 C L ._ I O

.F E - 0 0 r

0.01 0.1 1 10 100 1000

[Inhibitor] (pM)

FIG. 2. Dose-response curves for the effects of NPPB (A), furosemide (m), and ninumate (0) on the malaria-induced influx of choline at 22 "C. The extracellular solution contained 1 m~ choline, as well as 1 m~ thymidine (see Fig. 7). The induced flux was calculated by subtracting that measured in uninfected erythrocytes (cultured in parallel with infected cells) from that measured in infected cells from the same donor and is expressed as a percentage of the induced flux measured in the absence of inhibitor. The data comprising each curve were averaged from four different experiments on cells from different donors (mean S.E.). In these four experiments the unidirectional influx of choline, measured in synchronized trophozoite-infected cell suspensions at 38-93% parasitemia, had a mean value of 31 f 8 p ~ o Y ( l O ' ~ RBC.h), while in uninfected cells from the same donors the mean choline i d u x was 2.9 f 0.8 pmoY(1012 RBC.h).

port we went on to test the effect of these reagents on the transport of many of the other classes of compounds to which infected cells show increased permeability. Screening was carried out using the semi-quantitative hemolysis method that has been used widely in previous studies of the permeability of infected erythrocytes (Ginsburg et al., 1985). Fig. 3.4 shows time courses for the hemolysis of malaria-infected erythrocytes suspended in an iso-osmotic solution of choline chloride. In the absence of inhibitors the infected cells lysed with a lh5,, value (i.e. the inverse of the half-time for hemolysis) of around 0.032 min-l. Niflumate (100 p), furosemide (100 p), and NPPB (100 p) all protected against hemolysis, as might be expected if the hemolysis was due to influx of choline via the induced pathways characterized in Fig. 2.

The remaining panels of Fig. 3 show time courses for the hemolysis of malaria-infected erythrocytes suspended in iso-

3342 Induced Dunsport in Malaria-infected Erythrocytes

n

E E

E x

3

.- 0 X

W

.- v)

v) x 0

a,

I 0

-

E

(A) choline

0 60 120

(C) L-serine

0 30 60

(E) L-alanine

0 30 60

( G ) L-valine

0 30 60

Time

( 8 ) L-glutamine

0 30 60

(D) L-threonine '::m 0 0 30 60

(F) D-olonine

0 30 60

(H) L-methionine

0 30 60

(min) FIG. 3. Effect of NPPB, furosemide, and niflumate (each at a

concentration of 100 p) on time courses for the hemolysis of

choline chloride, (B) L-glutamine, (C) L-serine, (D) L-threonine, malaria-infected cells suspended in ieossmotic solutions of (A)

( E ) L-alanine, (F) malanine, ( G ) L-valine, and (H) L-methionine, all at 37 "C. For each substrate the data are averaged from between 3 and 6 different experiments on cells from different donors (mean * S.E.). Symbols: 0, control; A, NPPB; B, furosemide; 0, niflumate.

osmotic solutions of a variety of amino acids. The l / ~ 5 0 values in the absence of inhibitors were 0.056 min-l for L-glutamine, 0.080 min-l for L-serine, 0.11 min" for L-threonine, 0.30 min-l for L-alanine, 0.31 min-' for malanine, 0.55 min" for L-valine, and 1.15 min" for L-methionine. The time course for hemolysis of cells in iso-osmotic L-alanine was similar to that in iso-osmotic D-alanine which implies that the induced transport mechanism was not stereoselective. For all of the amino acids tested niflumate, furosemide, and NPPB caused a reduction in the rate of hemolysis, consistent with their having reduced the uptake of substrate through the induced transport route. In all cases 100 p~ NPPB provided almost full protection against hemolysis throughout the duration of the experiment. Furose- mide at 100 p~ was somewhat less potent and niflumate at 100 p~ slightly less effective. The order of efficacy of the three inhibitors was therefore the same as that seen for their inhibition of malaria-induced choline influx (Fig. 2).

Fig. 4.4 shows time courses for the influx of L-carnitine, another electroneutral zwitterion, into normal and malaria-infected erythrocytes (measured from ~-['~Clcarnitine uptake). There was no significant influx of L-carnitine into uninfected cells over a 4-min period (when presented at gn extracellular concentration of 1 m ~ ) . By contrast, L-carnitine was transported into malaria-infected erythrocytes via a pathway that was blocked by furosemide (100 p ~ ) . Fig. 4B shows the corre-

0 2 4 0 30 60

Time (min) Time (min) FIG. 4. A, time courses for the influx of L-carnitine, a zwitterion, into

uninfected erythrocytes (0) and into malaria-infected erythrocytes (56% parasitemia) in the presence (B) and absence (0) of furosemide (100 p d . The extracellular L-carnitine concentration was 1 m ~ . The data are from a single experiment and are representative of those obtained in three similar experiments using cells from different donors. B, effect of NPPB, furosemide, and niflumate (each at a concentration of 100 p ~ ) on time courses for the hemolysis of malaria-infected cells suspended in an iso-osmotic carnitine solution at 37 "C. The data are averaged from three different experiments on cells from different donors (mean S.E.). Symbols: 0, control; A, NPPB; B, furosemide; 0, niflumate.

sponding hemolysis experiment. Infected cells suspended in an iso-osmotic carnitine solution lysed with a l / ~ ~ ~ value of 0.030 min-l. Niflumate (100 p ~ ) , furosemide (100 p ~ ) , and NPPB (100 p ~ ) each provided full protection against hemolysis for up to 60 min.

Malaria-induced Sugar %nsport"As with the induced transport of amino acids, the malaria-induced transport of a variety of sugars was inhibited by niflumate, furosemide, and NPPB. Fig. 5 shows time courses for the hemolysis of malaria- infected erythrocytes suspended in iso-osmotic solutions of the 5-carbon sugar wribose, the 6-carbon sugars D-galactose and D-glucose, the 6-carbon sugar-alcohol D-sorbitol, and the disac- charide sucrose.

In the ribose solution the infected cells lysed with a l/~50 value of 0.78 min-l; the rate of hemolysis was reduced by niflumate, more so by furosemide, and more so by NPPB. Hemol- ysis in the 6-carbon sugar solutions was slower than in the ribose solution; the l/~50 values were 0.26 min" in the D-sorbitol solution, 0.10 min-l in the D-glucose solution, and 0.088 min-l in the D-galactose solution. In each of these solutions hemolysis was prevented almost completely during the time scale of the experiments by the three inhibitors, although infected cells in iso-osmotic sorbitol solution containing 100 p~ niflumate showed some hemolysis after 60 min. As has been shown previously (Ginsburg et al., 1983), malaria-infected cells did not lyse in an iso-osmotic sucrose solution.

Malaria-induced Nucleoside lkansport-A number of recent studies have focused on the enhanced permeability of malaria- infected erythrocytes to nucleosides (reviewed by Gero and Up- ston (1992)). We therefore tested the effects of the three inhibitors identified in this study on the transport into infected cells of the purine nucleoside adenosine and the pyrimidine nucleosides thymidine and uridine. Fig. 6 shows time courses for the uptake of (A) adenosine and ( B ) thymidine (each presented at an extracellular concentration of 1 m ~ ) by normal and malaria- infected erythrocytes. The cells were pretreated with 20 p~ NBMPR to inhibit the endogenous erythrocyte nucleoside transporter. For both adenosine and thymidine, influx into infected cells was markedly enhanced and was inhibited by niflumate, more so by furosemide and more so by NPPB.

Also shown in Fig. 6 are time-courses for hemolysis of infected cells suspended in iso-osmotic solutions of ( C ) thymidine

Induced ?Fansport in Malaria-infected Erythrocytes 3343

100 n (A) sucrose

50 1 I 0- 0 30 60

(E) D-galactose ( C ) D-glucose

0 30 60

(D) D-sorbitol

0 30 60

(E) D-ribose

0 30 60 0 6 12

Time (min) FIG. 5. Effect of NPPB, furosemide, and nifiumate (each at a

concentration of 100 p a ) on time courses for the hemolysis of malaria-infected cells suspended in iso-osmotic solutions of (A) sucrose, (B) wgalactose, (C) *glucose, ( D ) 0-sorbitol, and ( E ) *ribose, all at 37 "C. The glucose, galactose, and ribose solutions contained 20 p cytochalasin B to inhibit the native erythrocyte sugar transporter. The data are averaged from between three and six different experiments on cells from different donors (mean f S.E.). Symbols: 0, control; A, NPPB; ., furosemide; 0, niflumate.

and (D) uridine.3 Qualitatively, the pattern of inhibition is the same as that seen in the previous hemolysis experiments, the three inhibitors provided protection against hemolysis in the order niflumate < furosemide < NPPB. Quantitatively, it is clear from Fig. 6, C and D, that hemolysis of infected cells in iso-osmotic thymidine and undine solutions was less sensitive to inhibition than was hemolysis of infected cells in iso-osmotic solutions of the other classes of substrate tested (Figs. 3-5). For parasitized cells suspended in an iso-osmotic thymidine solution, niflumate and furosemide caused only a moderate reduction in the rate of hemolysis, and even in the presence of NPPB there was significant hemolysis after 12 min (Fig. 6C). One possible explanation for the variation in inhibitor potency in the different iso-osmotic solutions is that different classes of substrate use distinct transport pathways, all sensitive to inhibition by niflumate < furosemide < NPPB, but with different ICso values in each case. An alternative possibility is that the uptake is mediated by a single pathway whose inhibitor sensitivity vanes between the different iso-osmotic solutions, perhaps as a result the more hydrophobic substrates (such as thymidine and uridine) competing with the inhibitors for occu- pancy of the transport pathway when presented at the very high concentrations used in the hemolysis experiments (ie. 300 m). To distinguish between these possibilities we compared dose-

response curves for the effect of the three inhibitors on the influx of r3H1thymidine with those for their effect on the influx of [14C-]choline into malaria-infected cells under identical extracellular conditions. For these (paired) experiments, cells

other purine nucleosides as they are not sufficiently water-soluble to Hemolysis experiments could not be camed out with adenosine or

allow the preparation of iso-osmotic solutions.

Inf lux time-courses (A) adenosine (8) thymidine

40

0.0 0.1 0.2 0 2 4

Time (min)

Hoemolysis time-courses

( C ) thymidine (D) uridine

0 6 12 0 30 60

Time (min)

( B ) thymidine into uninfected erythrocytes (V) and into malaria-in- FIG. 6. Upperpanels, time courses for the influx of (A) adenosine and

feded erythrocytes (79% and 87% parasitemia, respectively) in the absence of inhibitors (0) and in the presence of niflumate (.), furosemide (W), or NPPB (A), each at a concentration of 100 w. The temperature was approximately 22 "C. All samples contained 20 p NBMPR to inhibit the native erythrocyte nucleoside transporter. The data shown are from a single experiment and are representative of those obtained in at least three similar experiments on cells from different donors. Lower panels, effect of NPPB, furosemide, and niflumate (each at a concentration of 100 p) on time courses for the hemolysis of malaria-infected cells suspended in iso-osmotic solutions of (C) thymidine and ( D ) uridine at 37 "C. The data are averaged from three different experiments

p NBMPR. Symbols: 0, control; A, NPPB; W, furosemide; 0, niflumate. on cells from different donors (mean f S.E.). All solutions contained 20

were suspended in physiological saline containing both 1 m thymidine and 1 lll~ choline (i.e. more than two orders of magnitude lower than the concentrations used in the hemolysis experiments). As is shown in Fig. 7, for each inhibitor, the dose-response curves for the two different substrates were very similar, and there was no significant difference (p > 0.15, paired t test) between the relative induced fluxes of the two substrates at any of the inhibitor concentrations tested. Thus, although the inhibitors were much less effective at protecting parasitized cells against hemolysis in an iso-osmotic thymidine solution than in an iso-osmotic choline solution, a quantitative comparison of the effect of the inhibitors on the flux of thymidine and choline into malaria-infected erythrocytes under the same extracellular conditions reveals no difference in the pharmacological sensitivity of the induced transport of these two substrates. This is consistent with the view that a single pathway mediates the malaria-induced transport of choline and thymidine and that the differences between the inhibitor sensitivities in the thymidine and choline hemolysis experiments are due to the different effects of very high concentrations of these two solutes on the pathway.

The kinetics of thymidine transport into malaria-infected erythrocytes was investigated, and the results are shown in Fig. 8. Furosemide-sensitive influx of thymidine into infected erythrocytes increased linearly with concentration up to 10 m, confirming the non-saturability of the furosemide-sensitive pathway over a concentration range far in excess of normal physiological values.

Malaria-induced Anion Bansport-Recently, two groups have reported a marked increase in the permeability of the malaria-infected erythrocyte to lactate (Kanaani and Ginsburg

3344 Induced Dansport in Malaria-infected Erythrocytes

0.01 0.1 1 10 100 1000

[Inhibitor] (pM)

FIG. 7. Dose-response curves for the effects of NPPB (A), furosemide (W), and niflumate (0) on the malaria-induced influx of thymidine (solid lines) at 22 "C. Cells were suspended in a standard HEPES-buffered saline containing 1 nm thymidine and 20 w NBMPR,

that measured in uninfected erythrocytes (cultured in parallel with as well as 1 m choline. The induced flux was calculated by subtracting

infected cells) from that measured in infected cells from the same donor. The data comprising each curve were averaged from five different experiments on cells from different donors (mean * S.E.). In these five experiments the unidirectional influx of thymidine, measured in syn-

had a mean value of 8.0 * 1.5 mm~l/( lO'~ RBC.h), while in uninfected chronized trophozoite-infected cell suspensions at 38-93% parasitemia,

cells from the same donors the mean thymidine influx was 0.59 e 0.09 mmol/(1012 RBC.h). Also shown (broken lines) are the corresponding

induced choline influx (taken from Fig. 2). For all three inhibitors there dose-response curves for the effect of the three inhibitors on malaria-

was, at each concentration, no significant difference (p > 0.15, paired t test) between the relative diminution of the induced thymidine and choline fluxes.

h I

a, c 73 .- .-

A

0 2 4 6 6 1 0

[Thymidinelo (mM)

infected erythrocytes (V, V) and malaria-infected erythrocytes FIG. 8. Concentration-dependence of thymidine influx into un-

(67% parasitemia; 0, W) in the presence (closed symbols) and absence (open symbols) of furosemide ( 5 0 0 p). The data shown are from a single experiment and are representative of that obtained in two similar experiments on cells from different donors.

(1991); Cranmer, Halestrap and Gutteridge, cited by Poole and Halestrap (1993)). Fig. 9A shows time courses for the transport of this monocarboxylate into infected and uninfected cells pretreated with DIDS (10 w, to inhibit the band 3 anion exchange mechanism) and p-chloromercuribenzene sulfonic acid (100 w, to inhibit the native erythrocyte monocarboxylate carrier; Deu- ticke et al. (1978)). Under these conditions there was very little influx of L-lactate into uninfected cells over a 2-min period. By contrast, L-lactate was taken up rapidly by malaria-infected cells via a pathway showing the same relative sensitivity to niflumate, furosemide, and NPPB as that seen for all other substrates.

Niflumate, furosemide, and NPPB are best known as inhibitors of C1- transport pathways (e.g. Cabantchik and Greger, 1992). We therefore investigated the transport of C1- into malaria-infected erythrocytes. The results are shown in Fig. 9B. In normal erythrocytes in which the band 3 anion exchanger was inhibited by pretreatment with 10 w DIDS there was negli-

(A) lactate (B) CI-

Y 4 0 1 2 0 2 4

Time ( m i n ) Time (min)

FIG. 9. A, time courses for the influx of lactate into uninfected erythrocytes (V) and into malaria-infected erythrocytes (81% parasitemia) in the absence of induced transport inhibitors (0) and in the presence of niflumate (O), furosemide (W), or NPPB (A), each at a concentration of 100 1.1~. The extracellular lactate concentration was 1 IIMI, and the temperature was approximately 22 "C. All samples contained 10 w DIDS and 100 wp-chloromercuribenzene sulfonic acid. B , time courses for the influx of C1- into uninfected erythrocytes (V) and malaria-infected erythrocytes (84% parasitemia) in the absence of induced transport inhibitors (0) and in the presence of niflumate (.), furosemide (W), or NPPB (A), each at a concentration of 100 w. The extracellular C1- concentration was 150 m, and the temperature was approximately 22 "C. All samples contained 10 w DIDS. For both substrates the data shown are from a single experiment, representative of that obtained in several similar experiments on cells from different donors.

gible uptake of 36Cl- over 4 min. By contrast, the transport of 36Cl- into malaria-infected cells pretreated with 10 w DIDS was extremely rapid; the anion equilibrated between the intra- and extracellular compartments within a few seconds, too rapidly to allow an accurate measure of the induced transport rate, via a pathway that was inhibited by niflumate, furosemide, and NPPB in the same order of potency as was observed for the malaria-induced transport of all other substrates tested.

The induced transport of C1- into malaria-infected cells was faster than that of any of the other substrates tested in this study. This is illustrated in Fig. 10 which shows average values (mean 2 S.E.) for the induced transport rates of the different substrates. The values are those either measured or estimated for an extracellular concentration of 1 m and a temperature of 22 "C.

DISCUSSION

A wide variety of low molecular weight solutes has been shown previously to permeate malaria-infected erythrocytes via high capacity, non-saturable pathways that are functionally different from the transporters of normal cells (reviewed by Cabantchik (1990), Ginsburg (1990), and Gem and Upston (1992)). In the present study we have carried out experiments to address the question of whether the phenomenon is mediated by a single type of pathway or whether there are heterogeneous, substrate-specific transport pathways involved. In our initial experiments a number of anion transport blockers were identified as potent inhibitors of the enhanced cation permeability of malaria-infected cells. Three of these, NPPB, furosemide, and niflumate, were subsequently tested for their effect on the malaria-induced transport of neutral amino acids, sugars, nucleosides, and anions, using a combination of iso- osmotic hemolysis and radioisotope flux techniques. For each class of substrate tested the induced permeability was inhibited by the three inhibitors in order of potency: NPPB > furosemide > niflumate. The relative efficacy of the inhibitors at blocking hemolysis varied somewhat for the different iso-osmotic solutions tested (Figs. 3-6). However, a quantitative comparison of the effect of the inhibitors on the induced influx of thymidine and choline, substrates showing quite different

Induced Dansport in Malaria-infected Erythrocytes 3345

U S t . c1- 7 lactate

thymidine .adenosine

X u 1c m I I

I

1 o3

1 o2

10’

FIG. 10. Relative magnitudes of the malaria-induced influx of the different substrates tested in this study. For each substrate the induced flux was calculated by subtracting that measured in uninfected erythrocytes (cultured in parallel with uninfected cells) from that measured in infected cells from the same donor. Division of the induced flux by the parasitemia provided an estimate from each experiment of the malaria-induced flux in cells at 100% parasitemia. The values shown

cellular concentration of 1 m~ and a temperature of 22 “C. For choline, are mean values (+ S.E.) of fluxes measured or estimated for an extra-

adenosine, thymidine, and lactate direct measurements of induced influx were made under these conditions (Figs. 2,6,7, and 9). For K+ and carnitine, influx measurements made at 37 “C were temperature-cor- reded to 22 “C assuming the same temperature dependence as that measured for the induced choline flux. K+ and C1- influx measurements were made at extracellular concentrations of 5 and 150 m ~ , respectively (Figs. 1 and 9); the corresponding value at 1 m~ was estimated assuming a linear concentration dependence of the induced flux for these

bition of the induced flux by 100 p~ bumetanide (Table I) which was substrates. The measured K+ influx was also corrected for partial inhi-

included in all K’ transport experiments to eliminate K+ flux via the erythrocyte NaKClz cotransporter. The C1- influx was too fast to allow an accurate estimate of the induced transport rate (Fig. 9) and the value shown is likely to be an underestimate.

transport rates and inhibitor sensitivities in iso-osmotic hemolysis experiments, revealed no difference in the pharmacological sensitivity of the pathways by which these two disparate solutes entered infected cells in physiological media (Fig. 7).

The simplest explanation consistent with the pharmacological data obtained in this study is that most of the induced transport of cations, anions, amino acids, monosaccharides, and nucleosides into malaria-infected erythrocytes occurs via a single type of pathway. The functional characteristics of the pathway, its broad specificity, its non-saturability, and its failure to distinguish between stereoisomers (L- and D-alanine), are quite unlike those of a conventional transporter but are those expected of a pore or channel (Ellory et al., 1988; Stein, 1990). For those substrates for which quantitative influx measurements were made the magnitude of the malaria-induced (inhibitor-sensitive) transport was in the order: C1- > lactate > thymidine, adenosine > carnitine > choline > K+ (Fig. 10). The pathway is therefore anion-selective.

Anion-selective channels are widespread in the membranes of eukaryotic cells. Their physiological functions are generally assumed to relate to their permeability to the inorganic C1- ion, and they are commonly referred to as “chloride channels.” The pharmacology of the induced pathway of malaria-infected erythrocytes is similar to that of chloride channels in other cell types. NPPB was identified originally as an inhibitor of C1- conductance in rabbit kidney (Wangemann et al., 1986), and it is, for many C1- channels, the most potent inhibitor yet identified. The IC50 of around 0.8 1.1~ obtained here for the inhibition by NPPB of malaria-induced choline transport suggests that it is the most potent inhibitor of malaria-induced transport reported to date and falls within the range found previously for C1- channels in other cells (Wangemann et al., 1986; Paulmichl

et al., 1992; Cabantchik and Greger, 1992). Furosemide, which has been shown previously to block the transport of adenosine into mouse erythrocytes infected with Plasmodium yoelii (Gati et aE., 1990), has been suggested to inhibit Ca2+-activated C1- channels in rat lacrimal glands (Evans et al . , 1986), and it has been reported that both furosemide and bumetanide block C1- channels in bovine kidney (Pope et al., 1991). Niflumate blocks Ca2+-activated C1- channels in Xenopus oocytes (White and Aylwin, 1990), and MK-196, which blocked malaria-induced cation transport (Table I), inhibits C1- conductance in frog skin (Nagel et al., 1985) as well as volume-activated C1- channels in Ehrlich ascites tumor cells (Aabin and Hoffmann, 1986). DIDS which, at a concentration of 100 p ~ , caused partial (40-50%) inhibition of the malaria-induced choline and 86Rb+ influx (Table I) has a similar effect (i.e. partial blockage at 100 p ~ ) on an anion-selective channel in human platelets (Manning and Williams, 1989), and it also inhibits C1- channels in a variety of epithelial cells (summarized by Greger (1990) and by Frizzell and Cliff (1992)) and in lymphocytes (Sarkadi et al., 1985).

As with the pharmacology, the substrate selectivity of the malaria-induced pathway bears a striking resemblance to that of chloride channels in other tissues. Although anion-selective, the induced pathway has a significant permeability to both inorganic and organic cations. This is a characteristic of many types of chloride channel (Franciolini and Petris, 1990): for example, the “background chloride channel” of hippocampal neurons is permeable to monovalent inorganic cations (Fran- ciolini and Nonner, 1987) and has an even greater permeability to the organic cation, Tris (Franciolini and Petris, 1992); the anion-selective channel from human platelets is permeable to K+ and to the much larger tetraethylammonium ion (Manning and Williams, 1989); similarly, the “Ca2+-activated C1- channel” of Xenopus oocytes is permeable not only to monovalent inorganic cations, but also to the divalent cation, Ca2+ (Younget al., 1984).

The similarities between the substrate selectivity of the malaria-induced pathway and chloride channels elsewhere also extend to the permeability pattern seen for organic solutes. A comparison of the rates of hemolysis of infected cells (Figs. 3-6) in iso-osmotic organic solute solutions suggests that the malaria-induced pathway shows a preference for more hydrophobic compounds. For the amino acids (as has been noted previously by Ginsburg et al. (1985) and by Ginsburg and Stein (1987b)), the presence of a (hydrophilic) hydroxyl group de- creased the apparent rate of transport via the malaria-induced pathways (cf. valine versus threonine, alanine versus serine; Fig. 3) whereas the presence of (hydrophobic) methyl groups enhanced uptake (cf. valine versus alanine, threonine versus serine; Fig. 3). Of all the amino acids tested, methionine, the least water-soluble, showed the highest rate of induced transport (Fig. 3H). A similar trend is seen for the pyrimidine nucleosides; thymidine, with one less hydroxyl group and one more methyl group than uridine, permeated the infected cell much more rapidly ( c f . Fig. 6, C and D ) . The observation that adenosine permeated the pathway (Fig. 6 A ) while sucrose, a similarly sized but much more hydrophilic molecule, did not (Fig. 5A is again consistent with the view that hydrophobicity is an important determinant of permeation rate.

A number of chloride channels have been shown to accommodate organic ions, including anionic amino acids, polyols such as gluconate, and monocarboxylate acids such as lactate (Pollard et al . , 1991; Valverde et al., 1992; Halm and Frizzell, 1992; Roy and Malo, 1992; Banderali and Roy, 1992; Rasola et al., 1992). There has been relatively little attention paid to the factors governing organic substrate selectivity; however, there is evidence that at least one such channel, the background C1- channel of hippocampal neurons, shares with the malaria-in-

3346 Induced nansport in Malaria-infected Erythrocytes

duced transport pathway a preference for more hydrophobic solutes. The permeability of the background C1- channel to benzoate is nearly three times that to the smaller acetate ion and almost twice that to C1- itself, suggesting that the transport of organic substrates via this channel involves hydrophobic interactions between the solutes and components of the channel (Franciolini and Nonner, 1987). The results obtained in the present study, and previously (Ginsburg et al., 1985) are consistent with the same being true of the malaria-induced pathway. Such interactions may also be important in the mechanism by which the inhibitors identified in this study (all of which contain hydrophobic groups) block the pathway.

Whether the functional similarities between the transport pathway characterized here and chloride channels elsewhere are based on a genuine structural resemblance remains to be established. So too does the location and the physiological function(s) of the induced pathway. In the conventional view of the malaria-infected red cell the intracellular parasite is enclosed within both the parasitophorous vacuole and erythrocyte membranes, with compounds entering and leaving the parasite via the erythrocyte cytoplasm. In this model the induced channel is presumed to be situated at the red cell surface where it may facilitate the rapid entry of nutrients and the rapid exit of metabolic wastes from the infected cell cytoplasm. An alternative view, for which evidence is accumulating, is that the intracellular parasite may actually have direct access to the extracellular solution (Bodhammer and Bahr, 1973; Elford et al., 1985; Pouvelle et al., 1991; Loyevsky et al., 19931, either via a “metabolic window” (Bodhammer and Bahr, 1973) or a “parasitophorous duct” (Pouvelle et al., 1991). This remains contro- versial (e.g. Sherman and Zidovetzki, 1992; Haldar and Uy- etake, 1992; Fujioka & Aikawa, 1993); however, it does raise the possibility that solutes might enter the parasite directly, without passing through the erythrocyte cytoplasm. The solute pathway characterized here may therefore be either in the parasite membrane (with access being provided via a duct) or located at a “window” formed at a point of contact between the parasite, parasitophorous vacuole, and erythrocyte membranes. In either case it might be involved in the trafficking of metabolites and catabolites directly between the parasite cytoplasm and the external medium, although the question then arises of how the parasite maintains its ionic and biochemical composition in the presence of this high capacity “leak” pathway.

Another alternative is that the non-saturable, high capacity pathway characterized here is in the erythrocyte membrane but is not primarily involved in the supply of nutrients to the parasite. Instead, it may play a “housekeeping“ role in the host erythrocyte, perhaps being involved in cell volume control. As has been pointed out by Ginsburg and co-workers (Zarchin et al. 1986; Ginsburg, 1988) the digestion of hemoglobin by the intracellular parasite generates large amounts of free amino acids which, if allowed to accumulate in the red cell cytoplasm, will cause osmotic swelling. The primary role of the niflumate-, furosemide-, and NPPB-sensitive channel may therefore be to mediate the net efflux of amino acids from the host cell, thereby preventing its premature hemolysis. In this context it is perhaps significant to note that the pathway shows marked functional similarities to a pathway in fish erythrocytes that is activated by cell swelling and which mediates the volume-regu- latory emux of amino acids (predominantly taurine) from these cells (Fincham et al., 1987; Kirk et al., 1992~). Like the channel induced in malaria-infected human erythrocytes, the volume- activated pathway in fish red cells accommodates a diverse range of substrates including amino acids, sugars, and nucleosides; it does not saturate with increasing substrate concentration (up to 19 m ~ ) ; it shows an apparent preference for more

hydrophobic solutes; and it is inhibited by a range of chloride channel blockers, including furosemide, niflumate, MK-196 and, most effectively, NPPB (Kirk et al., 1992~). The functional characteristics of the pathway in fish erythrocytes are those of a volume-activated chloride channel although, as with the malaria-induced pathway, the molecular identity of this system remains to be established.

In summary, the pharmacological data obtained in this study are consistent with the hypothesis that much of the induced, non-saturable transport of low molecular weight solutes into malaria-infected erythrocytes occurs via a single type of pathway. This pathway shows marked functional similarities to chloride channels in other cell types and is therefore unlikely to be a simple defect in the lipid bilayer or a consequence of general membrane damage. This is relevant not only to our understanding of mechanisms of solute trafficking in malaria- infected cells but to the possible physiological roles of chloride channels elsewhere. Although, among the solutes tested here, the malaria-induced pathway showed the highest permeability to C1-, it had a significant permeability to a wide variety of both charged and uncharged organic compounds, and it is these, rather than C1-, that are likely to be the physiologically relevant substrates. This raises the question of whether the same might be true of chloride channels in other tissues. As has been discussed, a number of chloride channels have been shown to accommodate organic ions (and indeed, in a t least one case, to show a pronounced preference for large hydrophobic substrates). However, there is little information available regard- ing the permeability of such channels to uncharged solutes (which are not amenable to study using electrophysiological techniques), and there is little recognition of a physiological role for the permeability of such channels to anything other than C1-. The data obtained in the present study focus attention on the possibility that anion-selective channels might pro- vide effective routes for the high capacity transport of low molecular weight organic substrates and, furthermore, that this might be an important aspect of their physiological function.

Acknowledgment-We thank Steven Culliford for skilled technical assistance in a number of the early experiments in this study.

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