I. INTRODUCTION

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Malaria is an infectious disease, caused by unicellular, protozoan parasites of the genus Plasmodium. There are an estimated 300-500 million cases of the disease, world-wide, each year, giving rise to an estimated 1.5-2.7 million deaths (323). Four species of plasmodia are infectious to humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. It is the first of these, P. falciparum, that is responsible for the vast majority of deaths from malaria.

During the course of its complex life cycle, the malaria parasite invades the red blood cells of its vertebrate host, resulting in the unusual situation of one eukaryotic cell (the metabolically voracious and biosynthetically active parasite) living inside another (the comparatively inert erythrocyte). It is this phase of the parasite's life cycle that gives rise to all of the clinical symptoms of malaria. The strategy of living inside the cells of its host helps the parasite evade the host's immune system. However, it does pose significant challenges to the invading organism. The interior of the host erythrocyte represents a highly unusual extracellular environment (231). The intracellular parasite is confronted with an extracellular milieu that has, at least initially, high concentrations of K⁺ and proteins, low levels of Na⁺, and only trace levels of Ca²⁺. The invading parasite must have mechanisms for maintaining its chemical composition and for obtaining from the host cell cytosol all of the nutrients that it requires for its survival, doing so in competition with the metabolic and biosynthetic machinery of the host. Furthermore, there must be mechanisms for eliminating metabolic wastes, both from within the parasite and from the host cell. As in other cells, these processes involve membrane transport mechanisms that control the flux of solutes across the membranes of the host cell and the intracellular parasite. It is these mechanisms that are the major focus of this review.

It has long been recognized that after malaria infection the parasitized erythrocyte undergoes marked alterations in its basic membrane transport properties (reviewed in Refs 39, 40, 80, 109, 116, 120, 125, 130, 183, 276, 288). The activity of a number of the endogenous transport systems is altered. Furthermore, there appear in the infected cell new permeation pathways (NPP) with properties quite unlike those of any of the endogenous red cell systems. These pathways are yet to be identified at a molecular level; however, their functional characteristics have been described in some detail. The transport properties of the "parasitophorous vacuole" membrane (PVM) in which the intracellular parasite is enclosed, the parasite plasma membrane (PPM), and the membranes of the various organelles within the parasite are less well characterized. Functional studies, both of intact infected erythrocytes and of parasites isolated from their host cells using a variety of techniques, have provided some information about the transport properties of the PPM and PVM. The application of genetic techniques has yielded sequences of malaria parasite proteins that are homologous to membrane transport proteins from other organisms. Many more such sequences are emerging from the systematic sequencing of the P. falciparum genome (28, 58, 104, 306). P. falciparum has 14 chromosomes. The recently published sequence of chromosomes 2 and 3 of P. falciparum include four and three sequences, respectively, of putative membrane transporters (28, 104). One of these (on chromosome 2) has been shown to transport hexose sugars (190a, 349a, 350). Another gene (on chromosome 14) has been shown to encode a nucleoside transporter (44, 241b). At the time of writing, however, these are the only examples of Plasmodium-encoded transporters for which both the protein sequence and detailed functional characteristics (e.g., substrate specificity, kinetics etc.) have been established.

The aim of this article is to review what is currently known about the membrane transport systems that mediate the flux of solutes between the intraerythrocytic malaria parasite and the plasma. The major focus is on the most virulent of the malaria parasites infectious to humans, P. falciparum, but with reference made to other parasite and host species where appropriate. Most of the work discussed has been carried out since 1976 when a culture system that enabled the in vitro cultivation of P. falciparum in human erythrocytes first became available (320). Earlier studies of membrane transport phenomena in parasitized erythrocytes from malaria-infected animals have been reviewed elsewhere (288).

Section II gives a brief (and far from comprehensive) outline of the intraerythrocytic phase of the parasite life cycle, concentrating on those features that are relevant to the subject of this review. Section III deals with methodological issues and discusses the advantages and shortcomings of the various techniques that have been applied to the study of membrane transport in the malaria-infected cell. Section IV deals with general aspects of membrane transport in the malaria-infected erythrocyte, focusing in particular on the important (and contentious) issue of compartmentalization in the parasitized cell, and its implications for the interpretation of transport data. In sections V-VIII, the general transport properties of the different membrane systems in the malaria-infected erythrocyte are discussed, while section IX focuses in more detail on the various classes of solute for which there is information available regarding their transport in the parasitized cell.

	II. THE INTRAERYTHROCYTIC PHASE OF THE MALARIA PARASITE LIFE CYCLE

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Malaria parasites enter their vertebrate host via the bite of an infected female Anopheles mosquito. They make their way first, via the bloodstream, to the liver where a single parasite (or "sporozoite" as it is then called) invades a liver cell. Once inside, it multiplies to produce thousands of "merozoites." The liver cell swells and, eventually, bursts, releasing the merozoites into the circulation, where they set about invading the red blood cells of their host.

The different stages of the asexual intraerythrocytic phase of the parasite life cycle are represented schematically in Figure 1. The malaria parasite gains entry into its prospective host erythrocyte by a process that leaves the intracellular parasite enclosed within a PVM (discussed in more detail in sect. VI). In the hours immediately after invasion (the so-called "ring" stage), the intracellular parasite seemingly lies dormant. However, from ~15 h postinvasion there is a progressive increase in metabolic and biosynthetic activity within the infected cell as the parasite enters the "trophozoite" stage. The malaria parasite has a single mitochondrion but lacks a functional citric acid cycle and is thought to be wholly reliant on glycolysis for its energy supply. As the parasite matures, the rate of glucose utilization and lactic acid production by the parasitized cell increases to up to 100 times the rate in the uninfected erythrocyte (248, 267, 333). The parasite endocytoses portions of the erythrocyte cytoplasm into "cytostomal vesicles" that fuse with the internal digestive or food vacuole membrane. Here the proteins of the host cell cytosol (predominantly hemoglobin) are digested to small peptides (165, 186, 266) that serve as a source of amino acids for the parasite. There is extensive synthesis of proteins, RNA, and DNA, a situation that contrasts markedly with that in normal erythrocytes which lack the ability to synthesize macromolecules. Parasite-derived proteins are expressed not only within the parasite but are exported to the parasitophorous vacuole, to the PVM, to the cytosol, cytoskeleton, and plasma membrane of the host cell, and perhaps beyond, into the extracellular medium.

View larger version (37K): [in this window] [in a new window]   Fig. 1. Schematic representation of the different stages of the asexual intraerythrocytic phase of the life cycle of the malaria parasite Plasmodium falciparum. This phase begins with the invasion of an erythrocyte by a merozoite (a). The parasite engulfs a portion of erythrocyte cytosol so that in section it appears as a thin ring, at which point it is referred to as being at the "ring" stage (b). The ring stage parasite grows to become a "trophozoite"; the erythrocyte loses its characteristic smooth biconcave discoid appearance, and small, electron-dense protrusions known as "knobs" appear on its surface (c). At the "schizont" stage, the parasite subdivides to produce 20-30 daughter merozoites (d), then, ~48 h after the initial invasion, the host erythrocyte bursts, releasing the merozoites (e), and a new cycle begins.

Concomitant with this dramatic increase in metabolic and biosynthetic activity, the parasite grows in size until, by 36 h postinvasion, it occupies approximately one-third of the total volume of the host cell (273). It remains enclosed within the PVM, which increases in size accordingly. At the same time, there appears in the erythrocyte cytosol a variety of tubular and vesicular membrane structures, thought to extend out from the PVM and variously referred to as the "tubovesicular membrane" (TVM; Ref. 87) or "tubovacuolar" (80) network. There are pronounced changes in the morphology of the infected cell, which is transformed from the smooth biconcave disk of the normal erythrocyte to an irregularly shaped cell, the surface of which becomes covered with a plethora of small electron-dense protrusions known as "knobs." The knobs are the site of localization of a number of parasite-derived proteins, including the products of the so-called var gene family (60). These proteins, known collectively as Pfemp1 (for Plasmodium falciparum erythrocyte membrane protein 1), are integral membrane proteins that play a central role in the dual phenomena of cytoadherence (i.e., binding of infected cells to the endothelial cells lining the capillaries of the brain and other organs, as well as to uninfected erythrocytes) and antigenic variation (16, 264, 268, 298, 307).

In addition to the insertion of new proteins into the red blood cell membrane (RBCM), there is a marked alteration of the composition and organization of the lipid phase of this membrane (159, 211, 281, 294, 353), as well as some rearrangement and modification of the endogenous red cell membrane proteins. The band 3 anion exchanger, which is the most abundant of the host cell integral membrane proteins, undergoes a decrease in mobility (317), and a proportion of the band 3 proteins are also truncated by proteolytic cleavage (54, 55, 290).

Approximately 40 h after the initial invasion, the late-stage trophozoite enters the "schizont" stage at which point it subdivides to produce 20-30 daughter merozoites. These are released at "schizogony" when the host cell finally ruptures, some 48 h after invasion. Each of the new generation of merozoites is capable of invading another erythrocyte, thereby continuing the cycle.

	III. METHODS

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A number of different experimental techniques have been applied to the study of membrane transport mechanisms in the malaria-infected erythrocyte. In this section these techniques are considered in detail and their advantages and limitations are discussed.

A.  Cell Preparations

1.  Malaria-infected erythrocytes

Until the late 1970s, the majority of investigations into the physiology and biochemistry of the malaria-infected erythrocyte were carried out using blood taken from animals (mice, rats, birds, monkeys) infected with one of the various different species of plasmodia that infect animals of different species. Laboratory studies of malaria were revolutionized in 1976 with the development of a method for the in vitro culture of P. falciparum (320, 321). This method, in combination with techniques to synchronize the parasites in culture to within a few hours (198), and to separate parasitized from nonparasitized cells, either by centrifugation on a Percoll density gradient or using a simple gelatin flotation technique (244), enables the production of synchronized suspensions of P. falciparum-infected human erythrocytes in the quantities necessary for physiological and biochemical studies.

A) DETERGENTS. The plant-derived detergents saponin and digitonin interact with cholesterol in cell membranes, thereby causing a fundamental disruption of the barrier properties of cholesterol-containing membranes (84, 284, 285, 297). Treatment of parasitized erythrocytes with either saponin (e.g., Refs. 8, 9, 273, 327) or digitonin (e.g., Ref. 63) renders the RBCM freely permeable to solutes as large as soluble proteins (e.g., hemoglobin), while leaving the intracellular parasite intact. There is evidence that, in addition to its effect on the RBCM, saponin also permeabilizes the PVM in which the intracellular parasite is enclosed (9).

B) OTHER BIOLOGICAL AGENTS. Complement, in conjunction with an appropriate antiserum, has been used to permeabilize the erythrocyte membranes of parasitized cells (322). Ginsburg and colleagues (124, 166, 167) have made effective use of Sendai virus for the same purpose. The Sendai virions induce the fusion of erythrocytes, causing, in the process, the permeabilization of the erythrocyte membrane to small solutes. As a result, the host cell undergoes colloid osmotic hemolysis, leaving the parasite intact within freely permeable erythrocyte ghosts.

Streptolysin O is a bacterial protein that forms pores of >30 nm in diameter (22). It has been used previously to study the transport of peptides across the endoplasmic reticulum membrane of mammalian cells (233) and has been shown recently by Lingelbach and colleagues to provide an effective means of permeabilizing the RBCM of malaria-infected erythrocytes (8, 9). The same group has also provided evidence that whereas the detergent saponin permeabilizes both the RBCM and the PVM, streptolysin O permeabilizes only the former, leaving the latter intact (9).

In a very recent study, Lauer et al. (199a) have reported that treatment of trophozoite-stage parasitized erythrocytes with the cholesterol-depleting agent methyl--cyclodextrin causes the release of parasites, free of the PVM. The parasites may be obtained in high yield (50-70% of parasites are released) and remain viable for up to 24 h. Parasites obtained in this way may offer the opportunity to study the physiological properties of the PPM without interference from the PVM.

C) OSMOTIC LYSIS. The appearance in the membrane of malaria-infected erythrocytes of NPP some hours after invasion (see sect. VC) forms the basis of a number of methods for the selective disruption of the host erythrocyte membrane. Suspension of trophozoite-infected cells in an isosmotic solution of compounds that permeate the NPP freely, but to which the normal erythrocyte has a limited permeability, leads to the selective lysis of infected cells (see sect. IIIC). If the PPM and/or PVM has a lower permeability to the permeating solute than the RBCM, or if the parasite is able to actively regulate its volume and thereby counteract any osmotic swelling, it emerges from this procedure unscathed.

A variation on this approach involves suspending trophozoite-infected erythrocytes in culture medium made hyperosmotic by the addition of a solute able to permeate the NPP in the erythrocyte membrane. On exposure to the hyperosmotic medium, the infected cell shrinks (in response to the increased extracellular osmolality), then recovers its volume as the permeant solute enters the cell. On return of the cells to an isosmotic saline, the osmolality of the host cell compartment is higher than that of the external medium, and it therefore swells and bursts. Providing that the intracellular parasite is less permeable to the added solute than the host red cell membrane and/or it is able to withstand a greater hyposmotic shock than its host cell, it remains intact.

Hoppe et al. (158) have used this approach, with sorbitol as the permeant solute, to isolate P. falciparum trophozoites from their host cells. Elford (79) has described a similar approach using di- and tripeptides. In the latter protocol, cells are exposed to a (slightly) hyperosmotic solution of di- and tripeptides then transferred back to an isosmotic saline, whereupon the parasitized cells lyse, releasing the intracellular parasite. Although the mechanism underlying the peptide-induced hemolysis has not been elucidated in detail, the likely explanation is, as above, an initial shrinkage then gradual volume recovery for the cells in the hyperosmotic peptide medium, followed by the osmotic lysis of the host cell compartment on return of the cells to isosmotic conditions. The use of peptides as the permeant solute in this procedure has the additional advantage that hydrolysis of the peptides (to their component amino acids) by peptidases within the host erythrocyte compartment (173) may increase the intracellular osmolality and thereby add to the magnitude of the osmotic shock to which the infected cells are exposed on return to isosmotic media.

D) PHYSICAL DISRUPTION. Nitrogen decompression of a malaria-infected cell suspension, involving exposure of the cells to a high pressure N₂ atmosphere (typically for 15 min), followed by their return to atmospheric conditions, results in the disintegration of the RBCM of parasitized erythrocytes into vesicles, leaving the majority of the parasites (as well as the majority of uninfected cells present) intact (237). Haldar et al. (142) have described the use of a stainless steel ball homogenizer to release intact parasites from their host erythrocytes. However, the yield of parasites from this method is relatively low (10-30%).

E) MEROZOITES AND AXENIC CULTURE OF PARASITES. An alternative approach to obtaining malaria parasites free of erythrocytes is to rely on the natural release of the parasites (merozoites) from their host erythrocyte at the end of each intraerythrocytic cycle (23). The merozoites would normally spend as short a time as possible in the extracellular medium before invading another erythrocyte (Fig. 1). They can, however, be harvested in sufficient quantity to allow biochemical and physiological measurements to be made (23, 327). In a recent study it was demonstrated that treatment of schizont stage parasites with a cysteine-protease inhibitor causes the accumulation in the medium of extraerythrocytic merozoites, trapped within the PVM (276a). These merozoites are viable and capable of normal erythrocyte invasion and development. They are readily purified from the medium and may therefore be used in the types of studies described in the following sections. Attempts to culture the erythrocytic stages of the malaria parasite extracellularly have shown that supplementation of the medium with erythrocyte extract permits the development of some of the parasites to the ring stage, although the yields are low (321).

This point is illustrated in Figure 2. Figure 2B shows an idealized time course for the uptake of a solute (denoted by S) that equilibrates rapidly between the erythrocyte cytosol compartment and the extracellular medium (Phase I) and is then either sequestered into the parasite, metabolized (to an impermeant form, denoted by S'), or both, at a much slower rate (Phase II). Under these conditions, uptake of radiolabel will provide a true measure of the transport of the solute across the RBCM only if it is measured over the very early portion of Phase I of the time course. The use of longer time periods that fall outside this initial linear phase will lead to an underestimate of the transport rate, as well as an overestimate of IC₅₀ values for inhibitors and of Michaelis constant (K_m) values for saturable transport processes.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Idealized time courses for the uptake of solute (denoted by S) into a multicompartmental system such as a malaria-infected erythrocyte, represented schematically in A. B: an idealized time course for the uptake of a solute that equilibrates rapidly across the erythrocyte membrane via a passive (i.e., nonconcentrative) process (Phase I) and that is then either metabolized (to an impermeant form; S'), sequestered into the parasite, or both, at a much slower rate (Phase II). C: an idealized time course for solute uptake under conditions in which the initial equilibration step (Phase I) is significantly slower than the subsequent step(s) (Phase II) so that the solute is sequestered and/or metabolized immediately on entering the parasitized cell. The "distribution ratio" is the total concentration of the solute (S + S') inside the cell, relative to that in the extracellular solution.

Figure 2C shows an idealized time course for solute uptake under conditions in which the initial transport step (Phase I) is significantly slower than the subsequent step(s) (Phase II), so that in practice, no sooner has a solute entered the host cell cytosol then it finds itself sequestered and/or metabolized. Under these conditions, the uptake of radiolabel may be rate-limited by the transport of solute across the RBCM for an extended period, during which the total concentration of radiolabel inside the cell may reach a much higher level than in the extracellular solution. This does offer significant advantages to the experimenter who, apart from anything else, will be able to use less radiolabeled substrate to make a quantitative estimate of the influx rate. However, it also holds significant dangers.

First, as discussed in general terms by Wohlhueter and Plagemann (349), if the concentration of metabolized or sequestered radiolabel is very large relative to the concentration of unaltered solute in the erythrocyte cytosol, then the uptake time course might appear to extrapolate through the origin, while not truly doing so (i.e., the time course may appear to take the form of Fig. 2C, whereas the real situation is actually that of Fig. 2B). This leads to an underestimate of the influx rate.

Second, even if under one set of conditions the rate of metabolism or sequestration (i.e., Phase II) is truly much greater than the rate of influx (Phase I), so that the rate of accumulation of radiolabel provides an accurate measure of the initial transport rate, this will not necessarily be the case under all conditions. If, in investigating the effects of different experimental conditions (e.g., increasing substrate concentration, addition of competitive substrates or of potential inhibitors), a particular maneuver reduces the rate of the metabolic or intracellular compartmentalization step (Phase II) while having a lesser effect on the initial transport step (Phase I), there is a risk that the compartmentalization process will become the rate-limiting process. In this case, the situation will revert to that represented in Figure 2B. If, under these conditions, the length of the uptake incubation falls outside the initial part of the time course, then the amount of radiolabel taken up will be affected by both the rate of transport and by the subsequent conversion or compartmentalization rate. In this case, the characteristics that emerge from such an analysis (kinetic constants, pharmacological properties) may be a combination of those of the transport step and those of the intracellular process(es).

An alternative method that has been used extensively to study the altered permeability of the malaria-infected erythrocyte, as well as various other induced-permeability phenomena in erythrocytes, involves suspending the cells in an isosmotic solution of the solute of interest. The principle behind this method is illustrated in Figure 3. On suspension of the cell in the isosmotic solution, there is a large inward concentration gradient, and hence a large driving force for the influx of the extracellular solute (represented by solid circles). If the permeability of the RBCM to this compound is higher than that to the solutes comprising the cell cytosol (represented by open circles), the rate of influx of material into the cell exceeds the rate of efflux, resulting in a net uptake of solute and water. This causes cell swelling and eventual hemolysis, the rate of which provides a semi-quantitative estimate of the (net) rate of influx of solute. Hemolysis is readily monitored by measuring the release of hemoglobin (spectrophotometrically, using absorbance at 540 nm), or that of other intracellular solutes (e.g., ATP; Refs. 40, 166).

View larger version (36K): [in this window] [in a new window]   Fig. 3. Schematic representation of the process by which parasitized erythrocytes suspended in an isosmotic solution of a permeant solute undergo "isosmotic hemolysis." The influx of extracellular solutes () at a rate greater than the efflux of cytosolic solutes () gives rise to a net uptake of solute and water, leading ultimately to hemolysis.

The isosmotic hemolysis technique has been used to investigate the permeability of the malaria-infected erythrocyte to a wide range of nonelectrolytes (127, 128, 179) and to a number of cations (178, 179, 303). It may be adapted for use with anions, although this requires that the permeability of the cell membrane to cations be higher than its permeability to the anions of interest. It is only under this condition that the net influx of the anion of interest is limited by the permeability of the anion itself, and not by the permeability of the accompanying cation (as would normally be the case). In practice, this can be achieved by the use of NH salts of the anions of interest (128). NH is not itself highly permeant but is in rapid equilibrium with NH₃, which traverses the membrane rapidly, thereby providing an effective means for NH to enter the cell (197).

The isosmotic hemolysis technique offers the major advantages of requiring relatively small amounts of material (the spectrophotometric determination of hemoglobin concentration is highly sensitive and allows the detection of the hemolysis of relatively few cells), of being applicable to infected cell suspensions at low parasitemia (uninfected cells are stable for long periods in isosmotic solutions of many of the solutes of interest and therefore do not contribute to measured hemoglobin release) and of not requiring the use of expensive radioisotopes.

However, it also has the following significant limitations.

1) Its application is restricted to solutes that are sufficiently hydrophilic to be soluble at the concentrations needed to make an isosmotic solution (i.e., ~300 mM for nonelectrolytes and ~150 mM for monovalent salts) and which are not hemolytic to normal erythrocytes at these high concentrations.

2) It requires that the cells be exposed to conditions that are far from physiological. This may affect the operation of the pathways of interest.

3) The technique provides information about the net influx of a particular solute under conditions in which the cell is exposed to a single, high concentration of that solute. If the influx pathway is saturated by high concentrations of the solute of interest, the rate of hemolysis will not be indicative of the true permeability of the pathway to the solute.

4) The rate of hemolysis is influenced not only by the net influx rate of extracellular solute but by the fate of the solute once it has entered the infected cell. In Figure 3, the solute is shown as being excluded from the intracellular parasite and remaining unaltered and in free solution within the erythrocyte cytosol. However, if the solute enters the parasite and is either metabolized or bound, in such a way as to change its osmotic contribution, then the amount of solute that will have to enter the cell to produce a given amount of cell swelling (and, ultimately, hemolysis) may be either more or less than if this does not occur. Under such circumstances, estimates of the relative permeation rates of different solutes from relative rates of hemolysis are, at best, semi-quantitative.

5) The technique is of limited use in comparing the permeation of different solutes (or the effects of inhibitors on the influx of different solutes) as the different isosmotic solutions provide quite different extracellular environments and the properties (e.g., inhibitor sensitivity; see Ref. 179) of the pathways of interest may well vary between these different conditions.

In summary, the isosmotic hemolysis technique provides a semi-quantitative measure of net solute permeation rates under limited (nonphysiological) conditions. It offers a convenient means for testing relative potencies of different inhibitors on the transport of any given substrate (albeit under extremely nonphysiological conditions). However, as noted in point 5 (above), caution must be exercised in using this approach to compare the effect of one or more given inhibitors on the transport of different substrates.

D.  Fluorescence

1.  Fluorescent transport solutes

Cabantchik, Ginsburg, and colleagues have used both the efflux (193, 195) and influx (34) of the fluorescent anion NBD-taurine to probe the altered permeability properties of the parasitized erythrocyte. In the efflux experiments, cells were preloaded with the fluorescent solute, washed, then suspended at low hematocrit in saline. The fluorescence of the suspension increased as the compound effluxed from the cells. This approach offers an advantage over analogous radiotracer experiments in allowing "on-line" measurements. However, it is restricted to fluorescent (and hence relatively large nonphysiological) substrates. It is also difficult to know with certainty which membrane in the infected cell constitutes the rate-limiting step for the efflux of the fluorescent probe that remains in the cell after the initial wash procedure.

More recently, larger fluorescent molecules such as Lucifer yellow (141, 199) and various fluorescent macromolecule conjugates (138, 153, 253) have been used in conjunction with fluorescence microscopy to study the uptake of such solutes into individual parasitized erythrocytes. The data are qualitative and, as discussed in section IVC, may, in some cases, be compromised by the dissociation of the fluorophore from the molecules of interest (153, 291).

Although fluorescent ion indicators have not, as yet, been widely applied to the study of the intracellular malaria parasite, there have been a number of recent studies demonstrating the applicability of this approach. Mikkelsen et al. (225) used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) to measure the intracellular pH (pH_i) of parasites (P. chabaudi) freed from their host cells using N₂ cavitation (see sect. IIIA2D), whereas Bosia et al. (27) used 6-carboxyfluoroescein to measure the pH_i of parasites (P. falciparum) within erythrocytes permeabilized using Sendai virus (see sect. IIIA2B). More recently, Wunsch and colleagues have described the use of BCECF in conjunction with a digital imaging system, to monitor the cytosolic pH of the parasite [both within intact erythrocytes and in parasites released from their host erythrocytes using the peptide hemolysis technique described in sect. IIIA2C; (355, 356)] and the pH in the cytoplasm of the host erythrocyte (see sect. IXJ). The same group has used the Na⁺-sensitive dye benzofuran isophthalate acetoxymethyl ester (SBFI) to monitor the concentration of Na⁺ within the intracellular parasite (see sect. IXJ) (354, 356).

Several other groups have reported the use of the fluorescent Ca²⁺ indicators indo 1, fluo 3 (1), and fura 2 (102) to estimate cytosolic Ca²⁺ concentrations in intact and/or permeabilized malaria-infected erythrocytes, as well as the use of the colourimetric Ca²⁺ indicator arsenazo III (242), in isolated parasites. The transport and homeostasis of Ca²⁺ in the malaria-infected erythrocyte is discussed in detail in section IXK.

Early estimates of the Na⁺/K⁺ composition of malaria-infected erythrocytes were made using flame photometry of extracts of erythrocytes from malaria-infected animals (74). These measurements did indicate a perturbation of the normal Na⁺/K⁺ balance in infected erythrocytes; however, the conclusions that could be drawn were limited by the multi-compartmental nature of the parasitized cell. Ginsburg et al. (124) used flame photometry, in combination with Sendai virus permeabilization of the host cell membrane (see sect. IIIA2B), to estimate the Na⁺/K⁺ concentration ratio in the host cell and parasite compartments of malaria-infected cells, showing it to be increased to well above normal levels in the red cell cytosol but maintained at a low level within the parasite. Similar results were obtained by Lee et al. (200) who used X-ray microanalysis in conjunction with electron microscopy to obtain estimates of the Na⁺, K⁺, Cl, and phosphorous content of the different compartments of the malaria-infected erythrocyte. The transport of monovalent inorganic cations in the parasitized erythrocyte is discussed in detail in section IXJ.

The patch-clamp technique involves the formation of a high-resistance (giga-ohm) seal between a cell membrane and a glass micropipette, then monitoring the currents arising from the flux of ions either across the enclosed patch of membrane or across the whole cell membrane (see Ref. 155). This technique has proven invaluable in elucidating the characteristics of ion channels in many animal and plant cells, but it has not, as yet, been widely applied to parasitic protozoa.

Patch-clamping malaria-infected erythrocytes is not straightforward. The infected cells are, compared with the cell types with which most electrophysiologists are familiar, both small and fragile, with a tendency to either burst or to disappear up into the patch pipette on application of suction. The earliest mention in the literature of patch-clamp data from intact, malaria-infected erythrocytes of which I am aware is in a review by Cabantchik (39) which refers to unpublished data (from Stutzin and Cabantchik) suggesting the presence of a voltage-dependent, phloridzin-sensitive ion channel in the infected cell membrane. However, the data were not presented.

In a study of Ca²⁺ transport in the malaria-infected erythrocyte, Desai et al. (64) reported a series of cell-attached patch-clamp measurements on intact parasitized erythrocytes. In these experiments they observed (in 2 of 26 parasitized cells tested) a seemingly novel channel activity. In each case, however, the cell lysed before the channel could be characterized in any detail (see sect. IXK). Very recently, Desai and colleagues (62a, 67) have reported obtaining both whole cell and cell-attached recordings of intact, trophozoite-stage parasitized erythrocytes and obtained evidence for a novel, voltage-dependent anion channel (see sect. VC3).

Desai et al. (63) have also described single-channel recordings from the PVM enclosing parasites freed from their host erythrocytes using two different techniques (digitonin and an electrical pulse applied to the host cell membrane). Similar recordings were obtained in a study in which the membrane fraction of homogenized intact parasitized erythrocytes were reconstituted into a planar lipid bilayer (65). The characteristics of this channel are discussed in section VIB.

The techniques of modern molecular biology have, over the past decade, yielded sequences of a number of putative plasmodial membrane transport proteins. In all cases, this has involved cloning homologs of transporters from other organisms. These include a number of P-type ATPases (75, 172, 188, 189, 324, 325), two V-type ATPase subunits (170, 171), several members of the ABC transporter family (29, 96, 347), and homologs of the mitochondrial ATP/ADP exchanger (76, 149, 150) and phosphate transporter (20).

The malaria genome sequencing project is now nearing completion (58, 104, 306). The recently published sequence of chromosomes 2 and 3 of P. falciparum include a total of seven putative transporter sequences (28, 104) and, as the genome sequencing project progresses, a wealth of other such sequences are becoming available. This poses a major challenge to those in the field. Functional expression of malaria-encoded membrane proteins is difficult, particularly if they are large (as is likely to be the case for many transporters and channels). The recent reports of increased transport of several solutes into Xenopus oocytes injected with P. falciparum mRNA (247) and the successful expression of cloned P. falciparum hexose (190, 190a, 349a, 350) and nucleoside (44, 241b) transporters indicate that the Xenopus oocyte is likely to be an extremely useful tool for the characterization of plasmodial transport proteins, as well as, perhaps, for the identification of novel transport proteins by expression cloning (247). However, the Xenopus oocyte system does have limitations, not least of which is the presence in these cells of an array of endogenous transporters and channels, some of which are activated in response to the expression of "foreign" proteins (e.g., Refs. 38, 292, 326).

The ability of at least one plasmodial ABC protein to complement a transport-deficient yeast strain (340) indicates that yeast might be a suitable system in which to clone (by complementation) and/or characterize plasmodial transporters and channels. This approach has proven highly successful in the identification and characterization of a range of transporters and channels from plants (98) but has not, as yet, been widely used in other organisms.

Other approaches still in their infancy in this field but which will, in the longer term, yield vital information regarding the function and physiological role(s) of the proteins of interest within the parasite include the use of antisense oligonucleotides (14, 15, 59, 257), ribozymes (93), gene knockout (53, 344), and gene transfection (334, 344, 352).

	IV. SOLUTE TRAFFICKING ROUTES IN THE PARASITIZED CELL

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According to the traditional view of the malaria-infected erythrocyte, represented in Figure 4A, the movement of solutes between the intracellular parasite and the external milieu occurs via the erythrocyte cytoplasm. Solutes taken up into the intracellular parasite have first to gain entry to the erythrocyte, across the RBCM. From here they can move into the parasite either by being transported sequentially across the PVM and PPM or by endocytosis (see sect. VII).

View larger version (26K): [in this window] [in a new window]   Fig. 4. Schematic representations of alternative solute trafficking routes in the malaria-infected erythrocyte. A: traditional view of the parasitized erythrocyte, in which solutes moving between the parasite and the extracellular medium do so via the erythrocyte cytosol, crossing the red blood cell membrane (RBCM), the parasitophorous vacuole membrane (PVM), and the parasite plasma membrane (PPM). This is referred to as the "sequential pathway." B and C: alternative "parallel pathways" that allow solutes to move between the parasite and the external medium without passing through the erythrocyte cytsosol. B shows different types of "metabolic window," specialized regions of membrane facilitating the exchange of solutes between the external medium and the parasite. At a, the PPM and PVM are closely apposed to the RBCM, as described by Bodammer and Bahr (24). At b, an extension of the so-called tubovesicular membrane (the TVM, extending out from the PVM) is fused with the RBCM to form a specialized junction, across which the exchange of solutes can take place, as postulated by Lauer et al. (199). C shows the proposed (highly contentious) parasitophorous duct, an open tubular structure that allows solutes in the external medium free access to the parasite surface (253).

In recent years there has been considerable interest in the possibility that there is, in addition to the "sequential route" (in which solutes cross each of the three membranes in sequence) outlined above, one or more additional "parallel routes" that allow solutes to move between the parasite and the external medium, without their actually entering the erythrocyte cytosol. There is evidence that the plasma membrane of the parasitized erythrocyte is incapable of endocytosis (143, 251), as is thought to be the case for the normal, mature erythrocyte. It remains controversial, however, whether there might be some means by which the parasite accesses the external medium other than via transport across the host erythrocyte membrane, into the red cell cytoplasm.

As long ago as 1973, Bodammer and Bahr (24) proposed, on the basis of scanning and transmission electron micrographs of P. berghei-infected mouse erythrocytes, that a localized region of apposition of the intracellular parasite to the red blood cell surface might serve as "a specialized entry and exit site for metabolites" and coined the phrase metabolic window (Fig. 4Ba). Lauer et al. (199) have recently proposed a variation of this model in which specialized regions of membrane formed at points of contact between the TVM and the RBCM serve as a route of entry for low-molecular-weight solutes into the TVM network, from where they are taken up by the parasite (Fig. 4Bb). However, much of the recent attention has focused on the proposal from Taraschi and colleagues (253) that the parasite has direct access to the extracellular solution via a so-called "parasitophorous duct," a tubular membranous structure that extends between the parasitophorous vacuole membrane and the erythrocyte membrane. The duct, as originally proposed, would allow the parasite plasma membrane to come into direct contact with the extracellular solution (Fig. 4C) and would provide a means for the intracellular parasite to take up macromolecules from the external medium, across the PPM, by a process of endocytosis. This proposal has been the subject of considerable controversy and in the heated debate surrounding the question of whether the duct exists, there has been a tendency for a number of related but separate issues to become intertwined. Here, two issues are considered separately. The first is the question of whether there is some form of parallel route that allows solutes to move between the intracellular parasite and the external solution, without actually entering the erythrocyte cytosol. The second is the question of whether the malaria-infected erythrocyte has the capacity to take up at least some macromolecules from the extracellular medium.

In two intriguing studies, Cabantchik and colleagues (209) showed that two different Fe³⁺ chelators (desferrioxamine and a fluorescent derivative thereof) and the bioflavonoid glycoside phloridzin (208) were toxic to the parasite when added to the extracellular solution, but had little effect on the parasite when they were encapsulated (at much higher concentrations) within red blood cells that were subsequently infected by the parasite. In interpreting these results, the authors proposed that these reagents cannot enter the parasite from the red cell cytosol but are able to do so only from the external solution (via some form of parallel route). This interpretation is consistent with the data; however, alternative explanations cannot be ruled out.

One possibility is that one or more of the agents tested exert their cytotoxic effects at the external surface of the infected cell, perhaps by blocking the uptake of nutrients and/or the release of metabolic wastes (80). Phloridzin does block the induced transport of small solutes into parasitized cells (194, 293). However, the same is not known to be true of the Fe³⁺ chelators, and there is evidence that desferrioxamine exerts its antiplasmodial effect from within the parasite (283).

Another possibility is that in the experiments with cells preloaded with the different antiplasmodial agents then invaded by the parasite, leakage of the compounds from the cytosol of the infected erythocytes into the extracellular medium reduced their concentration (both inside and outside the cell) to below that required to exert an antiplasmodial effect. Parasitized erythrocytes do have a substantially increased permeability to a wide range of solutes (sect. V), and Loyevsky and Cabantchik (208) demonstrated that erythrocytes preloaded with the different reagents did lose the majority to the external medium, particularly once the parasites reached the mature trophozoite-schizont stage (which is when the different drugs of interest exert their major antiplasmodial effect). It was argued that the concentration remaining within the infected cell should have been more than enough to retard parasite growth. However, it was not demonstrated that the drug retained by trophozoite-infected cells was actually in the erythrocyte cytosol. At least some may have been taken up into the parasite's food vacuole in the endocytotic feeding process (138, 319), before the induction of NPP in the RBCM and before the parasites become sensitive to the drug. Once there it may have been trapped, unable to gain access to potential targets elsewhere in the parasite.

A separate line of evidence for the existence of parallel routes comes from confocal microscopy studies of parasitized erythrocytes incubated with various fluorescent solutes, including several fluorescently labeled macromolecules and the smaller, widely used endocytosis marker Lucifer yellow. Papers describing a number of such studies report that fluorescence was localized to the intracellular parasite, and associated tubular structures in the host cell compartment, while apparently remaining excluded from the bulk host cell cytosol (138, 199, 252, 253). In the case of the fluorescently labeled macromolecules, concerns have been raised about dissociation of the fluorescent label (see sect. IVC). However, this issue notwithstanding, the question still arises of why in such experiments the fluorescence appears in the parasite and associated tubular structures, but not in the erythrocyte cytosol. The data have been interpreted as indicating that the fluorescent solutes are taken up directly into the parasite from the external medium (138, 252, 253). There are, however, a number of technical considerations, some or all of which may be relevant.

The composition of the red cell cytosol is quite different from that of the interior of the parasite and the TVM system, and it is possible that there is significant interference by components of the erythrocyte cytosol (in particular the hemoglobin) with fluorescent signal arising from this compartment. It is also possible that the fluorescent compounds are somehow accumulated within the parasite and the compartment(s) enclosed by the TVM, to levels substantially higher than those reached in the erythrocyte cytosol. Both situations would tend to give the appearance of there being negligible fluorescent compound in the host cell compartment, while not actually being the case.

Another possibility is that the lack of fluorescence associated with the host cell cytosol is due simply to the compounds leaking out of this compartment before (and perhaps during) the confocal microscopy measurements. In the majority of experiments of this sort, parasitized erythrocytes were preincubated for prolonged periods (typically 30-120 min) in the presence of fluorescent solute, then the "loading solution" was removed by washing the cells repeatedly before confocal measurements were made. It is conceivable that during the wash procedure, and subsequently, the fluorescent compound was lost from the host cell compartment, perhaps via NPP induced by the parasite in the host cell membrane (see sect. VC).

In summary, although there are several independent lines of evidence in support of the existence of parallel routes in the malaria-infected erythrocyte, none is entirely conclusive, and the issue awaits further clarification.

The existence of tubular structures traversing the cytosol of malaria-infected erythrocytes was described by Grellier et al. (139). However, it was Taraschi and colleagues (138, 253, 313) who first proposed that these tubes mediate the trafficking of macromolecules with diameters of up to 50-70 nm between the external medium and the parasite, and who coined the term parasitophorous duct (see Fig. 4C). This hypothesis was first proposed on the basis of experiments in which it was shown using confocal microscopy that macromolecules (e.g., fluorescent dextrans, biotinylated protein A, IgG antibody) and fluorescent latex beads, added to the extracellular medium, gained access to the aqueous space surrounding the parasite. In cells incubated with the fluorescent beads, fluorescence was shown to be associated with tubular structures that were proposed to connect the parasitophorous vacuole and host erythrocyte membranes.

The experiments of Pouvelle et al. (253) have been questioned on a number of technical grounds. Fujioka and Aikawa (99, 100) demonstrated that parasitized erythrocytes that had been maltreated in various ways took up colloidal gold and fluorescent dextrans, whereas parasitized cells maintained under normal conditions did not. This prompted the suggestion that the uptake of macromolecules described by Pouvelle et al. (253) was due to the parasitized erythrocytes used in this earlier study having been exposed to adverse conditions (99, 100), a contention strongly rejected by Taraschi and Pouvelle (313, 314).

Several others have emphasized potential problems arising from the dissociation of low-molecular-weight fluorophores from the fluorescently labeled probes used in the original study (143, 153, 291). In particular, Hibbs et al. (153), using a combination of confocal and electron microscopy, demonstrated that although incubation of malaria-infected erythrocytes with the fluorescent beads used in the original study by Taraschi and colleagues resulted in fluorescent labeling of the parasite and, in some cases, of associated tubular structures, the beads themselves (which had diameters down to 14 nm, well below that of the putative duct) remained excluded from the parasitized erythrocyte. The labeling of the parasite in this study was attributed to the release of membrane-permeant fluorescent dye from the beads during the incubation period, and it was suggested that the same phenomenon was responsible for the original results reported by Pouvelle et al. (253).

Using thin-layer chromatography, Goodyer et al. (138) demonstrated that the fluorescent dextrans used in the initial work of Pouvelle et al. (253) did undergo significant degradation during a 4-h incubation period. However, <0.0001% of the fluorophore molecules were released. It was argued that this could not account for the observed uptake of fluorescence by parasitized erythrocytes; however, it was not actually demonstrated that the fluorescence taken up into the intracellular parasite was in the form of the macromolecular dextran conjugate, and the data presented do not exclude the possibility that the fluorescence associated with the parasite is in the form of low-molecular-weight fluorophore molecules taken up from the external medium and perhaps concentrated from the extremely low levels in the extracellular solution to relatively high levels within the intracellular parasite.

Goodyer et al. (138) have also presented electron microscopic evidence for the uptake of ruthenium red, an electron-dense marker into ductlike structures that appeared to interconnect the erythrocyte membrane and the PVM. These findings would appear to be directly at odds with those of Elford and colleagues (80, 89), who have presented evidence that in parasitized erythrocytes exposed to ruthenium red, the compound remains entirely excluded from the infected cell. This is difficult to reconcile with the existence of a duct, as is the earlier finding by a number of groups (including that of Taraschi and colleagues) that parasitized erythrocytes fail to take up fluorescent molecules that have dimensions well below the diameter of the proposed duct (143, 251).

In addition to the various papers claiming to demonstrate directly the uptake of high-molecular-weight solutes into the malaria-infected erythrocyte (138, 252, 253), there are a number of studies that have been cited as providing independent evidence for the uptake by parasitized erythrocytes of at least some such solutes. These include a number of demonstrations that antisense oligodeoxynucleotides and ribozymes (i.e., oligonucleotides incorporating a sequence able to mediate the cleavage of complementary mRNA), targeted against parasite-encoded enzymes, inhibit the growth of the malaria parasite. Following on from the original reports of antisense oligonucleotides inhibiting parasite proliferation (59, 257), it was suggested that this was a nonspecific effect arising from the polyanionic oligonucleotides interfering with the invasion of the erythrocyte by the parasite (49, 256). It was shown subsequently, however, that although these reagents show sequence-independent effects when used at concentrations >1 µM, at lower concentrations their effects are sequence specific (14, 15). The same has also been shown to be true of ribozymes (93). The conclusion to be drawn from this work is that the oligonucleotides are somehow gaining access to the interior of the parasite.

In the original paper describing the antiplasmodial activity of antisense oligonucleotides, it was reported that radiolabeled antisense oligonucleotides were taken up by infected, but not normal, erythrocytes (257). However, the data were not presented, and it is not clear whether other explanations (e.g., uptake of radiolabeled products of oligonucleotide degradation) might account for the results described.

In a number of the studies of the antiplasmodial effect of antisense oligonucleotides, parasite growth was measured using asynchronous cultures and/or measured over a period that encompassed one or more schizogony and reinvasion steps. The data from these papers do not exclude the possibilities that the reagent(s): 1) targeted the merozoites during the brief period in between their release from one cell and invasion of another, 2) inhibited parasite invasion, or 3) entered the parasitophorous vacuole in sufficient quantity during the endocytotic invasion process to cause the subsequent retardation of parasite growth. However, in at least one study, oligonucleotides (ribozymes) were shown to exert a significant sequence-specific antiplasmodial effect within a single intraerythrocytic cycle (measured over 24 h after their addition to early ring-stage parasites; Ref. 93). In this case at least, there is therefore reason to believe that the oligonucleotides entered the parasitized erythrocyte at some time subsequent to the initial invasion step.

Oligonucleotides are not the only high-molecular-weight solutes reported to inhibit the growth of the intracellular malaria parasite. Gelonin, a single peptide chain protein inhibitor of protein synthesis, has been shown to inhibit parasite proliferation when exposed to parasitized erythrocytes for a fixed period within a single erythrocytic cycle (235). Dermaseptins, linear polycationic peptides composed of 28-34 amino acids, have also been shown to gain access to the intracellular malaria parasite within seconds of their addition to P. falciparum-infected human erythrocytes and to inhibit the growth of the parasite (114). The dermaseptins are amphipathic and do interact with lipid bilayers. Although it was argued that they do not translocate across the plasma membrane of normal uninfected erythrocytes, the data do not exclude the possibility that these compounds enter parasitized cells via the lipid phase of the RBCM.

Very recently it has been reported that addition to the culture medium of a 93-amino acid fragment of the enzyme -aminolevulinate dehydratase inhibits parasite growth (25a). It was shown using both immunofluorescence and a radiolabeled form of the polypeptide that the molecule (termed ALAD-NC) was taken up by infected but not uninfected cells. The radiolabel experiments provided evidence that the polypeptide was present within the parasite (including the food vacuole) but not in the erythrocyte cytosol, although the mechanism of uptake was not investigated.

There have also been reports that antibodies directed against antigens localized within the parasitized erythrocyte inhibit parasite growth (169). However, the mechanism by which they do so is unclear, and it has not been demonstrated that these antibodies are actually taken up into intact parasitized cells.

Table 1 provides a summary of the results of those studies that provide evidence in support of the view that the malaria-infected erythrocyte is able to take up macromolecules and other high-molecular-weight solutes from the external medium, as well as listing those which would argue against there being a nonspecific uptake of such solutes.

The two related questions of 1) whether there is a mechanism by which solutes can pass between the extracellular medium and the intracellular parasite without actually entering the erythrocyte cytosol, and 2) whether there is a mechanism by which the parasite is able to take up macromolecules and other high-molecular-weight solutes from the extracellular solution, have been, and remain, contentious. There is substantial evidence against the existence of a parasitophorous duct in the form originally proposed (253). Nevertheless, there is sufficient evidence in support of both hypotheses to warrant further investigation.

	V. THE RED BLOOD CELL MEMBRANE

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For some solutes there are a number of alternative transport pathways across the erythrocyte membrane, all of which may contribute to the measured influx or efflux. For example, the erythrocyte has at least four discrete and well-characterized K⁺ transport mechanisms (the Na⁺/K⁺ pump, the NaKCl₂ cotransporter, the KCl cotransporter, and the Ca²⁺-activated K⁺ channel) as well as others that are less well understood (e.g., Ref. 19). Amino acids are transported across the erythrocyte membrane via a number of different systems with overlapping specificity, e.g., at least five different pathways contribute to the flux of glycine across the erythrocyte membrane under physiological conditions (86). Similarly, the monovalent anion lactate permeates the membrane via at least three distinct pathways: a monocarboxylate carrier, the band 3 anion exchanger, and simple diffusion of the protonated acid across the bilayer (68, 250). In many cases, these alternative pathways can be distinguished on the basis of their different pharmacological and kinetic properties.

For any perturbation that causes an increase in the rate of transport across the erythrocyte membrane, the question arises of whether the increase is due to a change in the activity of endogenous systems or to the induction of new pathways. In the case of malaria infection (in which the parasite invades only a fraction of the erythrocytes available to it either in the bloodstream or in culture), the further question arises of whether an apparent increase in the flux via an endogenous pathway is due to a genuine change in the activity of that pathway in parasitized cells, or to the parasite invading preferentially a subpopulation of cells that have transport activity different from that of the population as a whole. Reticulocytes and young erythrocytes have higher activity of many transport systems than do mature erythrocytes (e.g., Refs. 145, 182). Thus, if the parasites have a significant preference for younger over older cells, the infected cells might be expected to show higher activity of many transporters than do uninfected cells in a suspension with a normal cell age distribution. Some strains of malaria (e.g., P. vivax) do show a very strong "reticulocyte preference" (226). P. falciparum shows a weak (2- to 3-fold) preference for reticulocytes over mature erythrocytes (226), although its relative preference for erythrocytes of different ages is not known.

Distinguishing membrane transport changes associated with altered flux via constitutively active endogenous systems from those arising from the insertion or activation of new pathways is not straightforward. In most cases the use of transport inhibitors, kinetic analyses, and (in some cases) different stereoisomers of relevant solutes enables the flux into or out of an infected cell to be dissected into functionally discrete components. The characteristics of these different components can be compared with those of the endogenous systems. However, the imperfect specificity of most inhibitors, as well as the possibility that the basic properties of the endogenous systems (e.g., pharmacology, substrate affinity) are fundamentally altered in the parasitized cell means that such analyses are rarely definitive. There is also the possibility (as yet neither proven nor excluded) that the parasite inserts into the erythrocyte membrane transport proteins having characteristics similar to those of the host cell.

Concerns such as those outlined above notwithstanding, there have been a number of studies showing enhanced transport in malaria-infected erythrocytes via pathways showing characteristics very similar to those endogenous to the host cell membrane. In human erythrocytes infected in vitro with P. falciparum, the activity of the Na⁺-K⁺ pump is increased by up to twofold (175, 300, 301a), probably due primarily to the raised Na⁺ concentration in the infected cell cytosol (see sect. IXJ). Ginsburg and Krugliak (126) found that in human erythrocytes infected in vitro with P. falciparum there was a significant increase in the maximum velocity (V_max) for the saturable component of tryptophan influx. The most striking examples of this phenomenon, however, come from experiments with erythrocytes taken from malaria-infected animals.

Parasitized erythrocytes from monkeys infected with P. knowlesi (4) and from mice infected with P. vinckei vinckei (302) both show increased uptake of choline via a pathway that has the same Michaelis constant (K_m) and pharmacological characteristics as the endogenous choline transporter, but a V_max some 10- to 20-fold higher than that in uninfected erythrocytes (see sect. IXF). By contrast, in human erythrocytes infected in vitro with P. falciparum, there is no evidence for an increase in flux via the endogenous choline transporter (82, 184).

Parasitized erythrocytes from P. knowlesi-infected monkeys also show an increased influx of the polyamine putrescine, via a saturable pathway with a K_m similar to that of the putrescine transporter of normal erythrocytes but a V_max some threefold higher than that seen in uninfected cells (295). It is possible that choline and putrescine share the same carrier (both are cations at physiological pH) and that the increase in the rate of transport of both substrates can be attributed to the increased activity of a single class of carrier. However, this has not been tested directly.

The mechanism underlying the increased rate of transport of substrates via pathways having the characteristics of endogenous host cell transporters remains to be clarified. After malaria infection, an erythrocyte undergoes many modifications of its physical/chemical properties, any of which might be expected to alter the activity of endogenous transport systems. The lipid composition of the erythrocyte membrane is altered (336), as are the cytoplasmic ion and, perhaps, protein concentrations. All of these are known to influence the activity of endogenous transporters and channels. Furthermore, the rate of influx and efflux of solutes via constitutive systems is affected by the cytoplasmic concentrations of the solutes themselves (via trans- as well as cis-effects). Alterations of these concentrations in the parasitized cell will therefore result in altered fluxes via the relevant transport systems.

In addition to causing an increased flux via pathways with the characteristics of endogenous host cell transporters, the intracellular malaria parasite induces in the host cell NPPs that have properties quite different from those of the endogenous transporters and which confer on the host cell an increased permeability to a wide range of solutes. The question of how many different types of NPP there are present in the parasitized erythrocyte has been addressed using a pharmacological approach. Various different classes of reagent have been shown to inhibit the NPP (see below). In experiments comparing the relative abilities of several of these reagents to inhibit the hemolysis of parasitized erythrocytes suspended in isosmotic solutions of different solutes (40, 41, 179), there was evidence that these reagents blocked the influx of some solutes with higher potency than that of others, prompting the suggestion that there are several different classes of parasite-induced pathway, each differing somewhat in its inhibitor sensitivity (40, 41). As discussed in section IIIC, however, the isosmotic hemolysis technique does have significant shortcomings and is not well-suited to comparisons of this type. More quantitative pharmacological studies, carried out by comparing the flux of radiolabeled solutes into cells suspended under identical conditions (which is not the case in the hemolysis experiments), have yielded data consistent with the view that the transport of a wide range of solutes occurs via common pathways. For each of a number of different inhibitors, the dose-response curves for the inhibition of the transport of several different structurally unrelated solutes are superimposable (177, 179, 327), consistent with (although not proof of) the hypothesis that much of the parasite-induced transport of small solutes into (and out of) the malaria-infected erythrocyte is mediated by NPP of a single type.

1) They are induced in the parasitized cell between 10 and 20 h postinvasion (301a).

2) They have a broad specificity and are permeable to a wide range of inorganic and organic monovalent ions (both cations and anions), zwitterions, and nonelectrolytes. It is unclear whether the NPP have a fixed size cut-off for permeating solutes. Although they have a low (it remains to be demonstrated whether negligible) permeability to sucrose (M_r = 342; Refs. 128, 179), there is evidence that they accommodate compounds as large as oxidized glutathione (GSSG; M_r&nbs