Membrane Transport in Malaria-Infected Erythrocytes (2001) PDF

PHYSIOLOGICAL REVIEWS Vol. 81, No. 2, April 2001 Printed in U.S.A. Membrane Transport in the Malaria-Infected Erythrocyte KIARAN KIRK Division of Biochemistry and Molecular Biology, Fac...

PHYSIOLOGICAL REVIEWS Vol. 81, No. 2, April 2001 Printed in U.S.A. Membrane Transport in the Malaria-Infected Erythrocyte KIARAN KIRK Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australian Capital Territory, Australia I. Introduction 496 II. The Intraerythrocytic Phase of the Malaria Parasite Life Cycle 497 III. Methods 498 A. Cell preparations 498 B. Radioisotope fluxes 499 C. Isosmotic hemolysis 501 D. Fluorescence 502 E. Ion analysis 503 F. Electrophysiological techniques 503 G. Genetic techniques 504 IV. Solute Trafficking Routes in the Parasitized Cell 504 A. Windows, tubes, vesicles, and ducts 504 B. Does the intracellular parasite have direct access to the extracellular medium? 505 C. Does the parasitized erythrocyte take up macromolecules and other high-molecular-weight solutes? 506 D. Summary 508 V. The Red Blood Cell Membrane 508 A. General considerations 508 B. Increased transport via pathways having the characteristics of endogenous host cell transporters 509 C. New permeation pathways 509 VI. The Parasitophorous Vacuole Membrane 515 A. Origin and composition of the PVM 515 B. Permeability properties of the PVM 515 VII. The Parasite Plasma Membrane 516 VIII. Intracellular Organellar Membranes 516 IX. Transport of Specific Solutes in the Parasitized Erythrocyte 517 A. Sugars 517 B. Amino acids 518 C. Peptides 518 D. Nucleosides 519 E. Vitamins 520 F. Choline 520 G. Lactate 521 H. ATP/ADP 521 I. Chloride 522 J. Sodium, potassium, and protons 522 K. Calcium 525 L. Magnesium 526 M. Drugs 526 X. Conclusions 528 Kirk, Kiaran. Membrane Transport in the Malaria-Infected Erythrocyte. Physiol Rev 81: 495–537, 2001.—The malaria parasite is a unicellular eukaryotic organism which, during the course of its complex life cycle, invades the red blood cells of its vertebrate host. As it grows and multiplies within its host blood cell, the parasite modifies the membrane permeability and cytosolic composition of the host cell. The intracellular parasite is enclosed within a so-called parasitophorous vacuolar membrane, tubular extensions of which radiate out into the host cell compart- ment. Like all eukaryote cells, the parasite has at its surface a plasma membrane, as well as having a variety of http://physrev.physiology.org 0031-9333/01 $15.00 Copyright © 2001 the American Physiological Society 495 Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 496 KIARAN KIRK Volume 81 internal membrane-bound organelles that perform a range of functions. This review focuses on the transport properties of the different membranes of the malaria-infected erythrocyte, as well as on the role played by the various membrane transport systems in the uptake of solutes from the extracellular medium, the disposal of metabolic wastes, and the origin and maintenance of electrochemical ion gradients. Such systems are of consider- able interest from the point of view of antimalarial chemotherapy, both as drug targets in their own right and as routes for targeting cytotoxic agents into the intracellular parasite. I. INTRODUCTION molecular level; however, their functional characteristics have been described in some detail. The transport prop- Malaria is an infectious disease, caused by unicellu- erties of the “parasitophorous vacuole” membrane (PVM) lar, protozoan parasites of the genus Plasmodium. There in which the intracellular parasite is enclosed, the para- are an estimated 300 –500 million cases of the disease, site plasma membrane (PPM), and the membranes of the world-wide, each year, giving rise to an estimated 1.5–2.7 various organelles within the parasite are less well char- million deaths (323). Four species of plasmodia are infec- acterized. Functional studies, both of intact infected tious to humans: Plasmodium falciparum, Plasmodium erythrocytes and of parasites isolated from their host cells vivax, Plasmodium malariae, and Plasmodium ovale. It using a variety of techniques, have provided some infor- is the first of these, P. falciparum, that is responsible for mation about the transport properties of the PPM and the vast majority of deaths from malaria. PVM. The application of genetic techniques has yielded During the course of its complex life cycle, the ma- sequences of malaria parasite proteins that are homolo- laria parasite invades the red blood cells of its vertebrate gous to membrane transport proteins from other organ- host, resulting in the unusual situation of one eukaryotic isms. Many more such sequences are emerging from the cell (the metabolically voracious and biosynthetically ac- systematic sequencing of the P. falciparum genome (28, tive parasite) living inside another (the comparatively 58, 104, 306). P. falciparum has 14 chromosomes. The inert erythrocyte). It is this phase of the parasite’s life recently published sequence of chromosomes 2 and 3 of cycle that gives rise to all of the clinical symptoms of P. falciparum include four and three sequences, respec- malaria. The strategy of living inside the cells of its host tively, of putative membrane transporters (28, 104). One helps the parasite evade the host’s immune system. How- of these (on chromosome 2) has been shown to transport ever, it does pose significant challenges to the invading hexose sugars (190a, 349a, 350). Another gene (on chro- organism. The interior of the host erythrocyte represents mosome 14) has been shown to encode a nucleoside a highly unusual extracellular environment (231). The transporter (44, 241b). At the time of writing, however, intracellular parasite is confronted with an extracellular these are the only examples of Plasmodium-encoded milieu that has, at least initially, high concentrations of transporters for which both the protein sequence and K⫹ and proteins, low levels of Na⫹, and only trace levels detailed functional characteristics (e.g., substrate speci- of Ca2⫹. The invading parasite must have mechanisms for ficity, kinetics etc.) have been established. maintaining its chemical composition and for obtaining The aim of this article is to review what is currently from the host cell cytosol all of the nutrients that it known about the membrane transport systems that me- requires for its survival, doing so in competition with the diate the flux of solutes between the intraerythrocytic metabolic and biosynthetic machinery of the host. Fur- malaria parasite and the plasma. The major focus is on the thermore, there must be mechanisms for eliminating met- most virulent of the malaria parasites infectious to hu- abolic wastes, both from within the parasite and from the mans, P. falciparum, but with reference made to other host cell. As in other cells, these processes involve mem- parasite and host species where appropriate. Most of the brane transport mechanisms that control the flux of sol- work discussed has been carried out since 1976 when a utes across the membranes of the host cell and the intra- culture system that enabled the in vitro cultivation of cellular parasite. It is these mechanisms that are the P. falciparum in human erythrocytes first became avail- major focus of this review. able (320). Earlier studies of membrane transport phe- It has long been recognized that after malaria infec- nomena in parasitized erythrocytes from malaria-infected tion the parasitized erythrocyte undergoes marked alter- animals have been reviewed elsewhere (288). ations in its basic membrane transport properties (re- Section II gives a brief (and far from comprehensive) viewed in Refs 39, 40, 80, 109, 116, 120, 125, 130, 183, 276, outline of the intraerythrocytic phase of the parasite life 288). The activity of a number of the endogenous trans- cycle, concentrating on those features that are relevant to port systems is altered. Furthermore, there appear in the the subject of this review. Section III deals with method- infected cell new permeation pathways (NPP) with prop- ological issues and discusses the advantages and short- erties quite unlike those of any of the endogenous red cell comings of the various techniques that have been applied systems. These pathways are yet to be identified at a to the study of membrane transport in the malaria-in- Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 497 fected cell. Section IV deals with general aspects of mem- more detail in sect. VI). In the hours immediately after brane transport in the malaria-infected erythrocyte, focus- invasion (the so-called “ring” stage), the intracellular par- ing in particular on the important (and contentious) issue asite seemingly lies dormant. However, from ⬃15 h post- of compartmentalization in the parasitized cell, and its invasion there is a progressive increase in metabolic and implications for the interpretation of transport data. In biosynthetic activity within the infected cell as the para- sections V–VIII, the general transport properties of the site enters the “trophozoite” stage. The malaria parasite different membrane systems in the malaria-infected eryth- has a single mitochondrion but lacks a functional citric rocyte are discussed, while section IX focuses in more acid cycle and is thought to be wholly reliant on glycolysis detail on the various classes of solute for which there is for its energy supply. As the parasite matures, the rate of information available regarding their transport in the par- glucose utilization and lactic acid production by the par- asitized cell. asitized cell increases to up to 100 times the rate in the uninfected erythrocyte (248, 267, 333). The parasite endo- II. THE INTRAERYTHROCYTIC PHASE OF THE cytoses portions of the erythrocyte cytoplasm into “cyto- MALARIA PARASITE LIFE CYCLE stomal vesicles” that fuse with the internal digestive or food vacuole membrane. Here the proteins of the host cell Malaria parasites enter their vertebrate host via the cytosol (predominantly hemoglobin) are digested to small bite of an infected female Anopheles mosquito. They peptides (165, 186, 266) that serve as a source of amino make their way first, via the bloodstream, to the liver acids for the parasite. There is extensive synthesis of where a single parasite (or “sporozoite” as it is then proteins, RNA, and DNA, a situation that contrasts mark- called) invades a liver cell. Once inside, it multiplies to edly with that in normal erythrocytes which lack the produce thousands of “merozoites.” The liver cell swells ability to synthesize macromolecules. Parasite-derived and, eventually, bursts, releasing the merozoites into the proteins are expressed not only within the parasite but circulation, where they set about invading the red blood are exported to the parasitophorous vacuole, to the PVM, cells of their host. to the cytosol, cytoskeleton, and plasma membrane of the The different stages of the asexual intraerythrocytic host cell, and perhaps beyond, into the extracellular me- phase of the parasite life cycle are represented schemat- dium. ically in Figure 1. The malaria parasite gains entry into its Concomitant with this dramatic increase in meta- prospective host erythrocyte by a process that leaves the bolic and biosynthetic activity, the parasite grows in size intracellular parasite enclosed within a PVM (discussed in until, by 36 h postinvasion, it occupies approximately FIG. 1. Schematic representation of the dif- ferent 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 mero- zoite (a). The parasite engulfs a portion of eryth- rocyte 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 erythro- cyte loses its characteristic smooth biconcave discoid appearance, and small, electron-dense protrusions known as “knobs” appear on its sur- face (c). At the “schizont” stage, the parasite subdivides to produce 20 –30 daughter merozo- ites (d), then, ⬃48 h after the initial invasion, the host erythrocyte bursts, releasing the merozo- ites (e), and a new cycle begins. Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 498 KIARAN KIRK Volume 81 one-third of the total volume of the host cell (273). It fected erythrocyte were carried out using blood taken remains enclosed within the PVM, which increases in size from animals (mice, rats, birds, monkeys) infected with accordingly. At the same time, there appears in the eryth- one of the various different species of plasmodia that rocyte cytosol a variety of tubular and vesicular mem- infect animals of different species. Laboratory studies of brane structures, thought to extend out from the PVM and malaria were revolutionized in 1976 with the development variously referred to as the “tubovesicular membrane” of a method for the in vitro culture of P. falciparum (320, (TVM; Ref. 87) or “tubovacuolar” (80) network. There are 321). This method, in combination with techniques to pronounced changes in the morphology of the infected synchronize the parasites in culture to within a few hours cell, which is transformed from the smooth biconcave (198), and to separate parasitized from nonparasitized disk of the normal erythrocyte to an irregularly shaped cells, either by centrifugation on a Percoll density gradi- cell, the surface of which becomes covered with a pleth- ent or using a simple gelatin flotation technique (244), ora of small electron-dense protrusions known as enables the production of synchronized suspensions of “knobs.” The knobs are the site of localization of a num- P. falciparum-infected human erythrocytes in the quan- ber of parasite-derived proteins, including the products of tities necessary for physiological and biochemical studies. the so-called var gene family (60). These proteins, known collectively as Pfemp1 (for Plasmodium falciparum 2. “Isolated” malaria parasites erythrocyte membrane protein 1), are integral membrane proteins that play a central role in the dual phenomena of Studies of the transport properties of the membranes cytoadherence (i.e., binding of infected cells to the endo- at the surface of the intracellular malaria parasite have thelial cells lining the capillaries of the brain and other entailed the use of a range of techniques to either free the organs, as well as to uninfected erythrocytes) and anti- parasite from its host erythrocyte or to permeabilize the genic variation (16, 264, 268, 298, 307). host cell membrane. The following approaches have been In addition to the insertion of new proteins into the used for this purpose. red blood cell membrane (RBCM), there is a marked A) DETERGENTS. The plant-derived detergents saponin alteration of the composition and organization of the lipid and digitonin interact with cholesterol in cell membranes, phase of this membrane (159, 211, 281, 294, 353), as well thereby causing a fundamental disruption of the barrier as some rearrangement and modification of the endoge- properties of cholesterol-containing membranes (84, 284, nous red cell membrane proteins. The band 3 anion ex- 285, 297). Treatment of parasitized erythrocytes with ei- changer, which is the most abundant of the host cell ther saponin (e.g., Refs. 8, 9, 273, 327) or digitonin (e.g., integral membrane proteins, undergoes a decrease in mo- Ref. 63) renders the RBCM freely permeable to solutes as bility (317), and a proportion of the band 3 proteins are large as soluble proteins (e.g., hemoglobin), while leaving also truncated by proteolytic cleavage (54, 55, 290). the intracellular parasite intact. There is evidence that, in Approximately 40 h after the initial invasion, the late-stage trophozoite enters the “schizont” stage at which addition to its effect on the RBCM, saponin also perme- point it subdivides to produce 20 –30 daughter merozoites. abilizes the PVM in which the intracellular parasite is These are released at “schizogony” when the host cell enclosed (9). B) OTHER BIOLOGICAL AGENTS. Complement, in conjunc- finally ruptures, some 48 h after invasion. Each of the new generation of merozoites is capable of invading another tion with an appropriate antiserum, has been used to erythrocyte, thereby continuing the cycle. 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. III. METHODS The Sendai virions induce the fusion of erythrocytes, causing, in the process, the permeabilization of the eryth- A number of different experimental techniques have rocyte membrane to small solutes. As a result, the host been applied to the study of membrane transport mecha- cell undergoes colloid osmotic hemolysis, leaving the par- nisms in the malaria-infected erythrocyte. In this section asite intact within freely permeable erythrocyte ghosts. these techniques are considered in detail and their advan- Streptolysin O is a bacterial protein that forms pores tages and limitations are discussed. of ⬎30 nm in diameter (22). It has been used previously to study the transport of peptides across the endoplasmic A. Cell Preparations 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 1. Malaria-infected erythrocytes malaria-infected erythrocytes (8, 9). The same group has Until the late 1970s, the majority of investigations also provided evidence that whereas the detergent sapo- into the physiology and biochemistry of the malaria-in- nin permeabilizes both the RBCM and the PVM, streptol- Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 499 ysin O permeabilizes only the former, leaving the latter host erythrocyte compartment (173) may increase the intact (9). intracellular osmolality and thereby add to the magnitude In a very recent study, Lauer et al. (199a) have re- of the osmotic shock to which the infected cells are ported that treatment of trophozoite-stage parasitized exposed on return to isosmotic media. erythrocytes with the cholesterol-depleting agent methyl- D) PHYSICAL DISRUPTION. Nitrogen decompression of a ␤-cyclodextrin causes the release of parasites, free of the malaria-infected cell suspension, involving exposure of PVM. The parasites may be obtained in high yield (50-70% the cells to a high pressure N2 atmosphere (typically for of parasites are released) and remain viable for up to 24 h. 15 min), followed by their return to atmospheric condi- Parasites obtained in this way may offer the opportunity tions, results in the disintegration of the RBCM of para- to study the physiological properties of the PPM without sitized erythrocytes into vesicles, leaving the majority of interference from the PVM. the parasites (as well as the majority of uninfected cells C) OSMOTIC LYSIS. The appearance in the membrane of present) intact (237). Haldar et al. (142) have described malaria-infected erythrocytes of NPP some hours after the use of a stainless steel ball homogenizer to release invasion (see sect. VC ) forms the basis of a number of intact parasites from their host erythrocytes. However, methods for the selective disruption of the host erythro- the yield of parasites from this method is relatively low cyte membrane. Suspension of trophozoite-infected cells (10 –30%). in an isosmotic solution of compounds that permeate the E) MEROZOITES AND AXENIC CULTURE OF PARASITES. An alter- NPP freely, but to which the normal erythrocyte has a native approach to obtaining malaria parasites free of limited permeability, leads to the selective lysis of in- erythrocytes is to rely on the natural release of the para- fected cells (see sect. IIIC). If the PPM and/or PVM has a sites (merozoites) from their host erythrocyte at the end lower permeability to the permeating solute than the of each intraerythrocytic cycle (23). The merozoites RBCM, or if the parasite is able to actively regulate its would normally spend as short a time as possible in the volume and thereby counteract any osmotic swelling, it extracellular medium before invading another erythro- emerges from this procedure unscathed. cyte (Fig. 1). They can, however, be harvested in sufficient A variation on this approach involves suspending quantity to allow biochemical and physiological measure- trophozoite-infected erythrocytes in culture medium ments to be made (23, 327). In a recent study it was made hyperosmotic by the addition of a solute able to demonstrated that treatment of schizont stage parasites permeate the NPP in the erythrocyte membrane. On ex- with a cysteine-protease inhibitor causes the accumula- posure to the hyperosmotic medium, the infected cell tion in the medium of extraerythrocytic merozoites, shrinks (in response to the increased extracellular osmo- trapped within the PVM (276a). These merozoites are lality), then recovers its volume as the permeant solute viable and capable of normal erythrocyte invasion and enters the cell. On return of the cells to an isosmotic development. They are readily purified from the medium saline, the osmolality of the host cell compartment is and may therefore be used in the types of studies de- higher than that of the external medium, and it therefore scribed in the following sections. Attempts to culture the swells and bursts. Providing that the intracellular parasite erythrocytic stages of the malaria parasite extracellularly is less permeable to the added solute than the host red have shown that supplementation of the medium with cell membrane and/or it is able to withstand a greater erythrocyte extract permits the development of some of hyposmotic shock than its host cell, it remains intact. the parasites to the ring stage, although the yields are Hoppe et al. (158) have used this approach, with low (321). sorbitol as the permeant solute, to isolate P. falciparum trophozoites from their host cells. Elford (79) has de- scribed a similar approach using di- and tripeptides. In the B. Radioisotope Fluxes latter protocol, cells are exposed to a (slightly) hyperos- motic solution of di- and tripeptides then transferred back Quantitative estimates of membrane transport rates, to an isosmotic saline, whereupon the parasitized cells as well as the investigation of the kinetic and pharmaco- lyse, releasing the intracellular parasite. Although the logical characteristics of membrane transport mecha- mechanism underlying the peptide-induced hemolysis has nisms in both intact malaria-infected erythrocytes and not been elucidated in detail, the likely explanation is, as isolated malaria parasites, have usually involved measur- above, an initial shrinkage then gradual volume recovery ing the influx (and, less often, the efflux) of radiolabeled for the cells in the hyperosmotic peptide medium, fol- forms of the solutes of interest. The general approach in lowed by the osmotic lysis of the host cell compartment influx experiments is to combine cells and radiolabeled on return of the cells to isosmotic conditions. The use of substrate, incubate them for an appropriate time, separate peptides as the permeant solute in this procedure has the the cells from the suspending medium (either by centri- additional advantage that hydrolysis of the peptides (to fuging the cells through an oil layer of density intermedi- their component amino acids) by peptidases within the ate between the cells and the aqueous solution or by Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 500 KIARAN KIRK Volume 81 repeated washing of the cells by centrifugation and resus- from the external medium as measured in initial rate pension in a “stopping solution”), then analyze the radio- experiments may occur across the host erythrocyte mem- activity in the cell pellet. A common strategy in such brane, the parasite membrane, or both. studies is to carry out initial time course experiments to Even if there are no such “parallel routes,” and the establish the period for which the uptake of solute re- traffic of all solutes between the parasite and the external mains approximately linear with time, then, in subsequent medium is via the erythrocyte cytosol, the issues of intra- experiments, to estimate influx rates from the amount of cellular compartmentalization and metabolism still raise radiolabel taken up during a fixed-length incubation that significant difficulties. If a solute, on entering the eryth- falls within this period. rocyte cytosol, is transported into the parasite and/or The intention in such experiments is usually to esti- metabolized, the question immediately arises of the ex- mate the initial rate of influx of radiolabeled substrate tent to which these processes determine the uptake of the into the cells. The major underlying difficulty with this solute of interest over the time period over which the approach is that it is not always a straightforward matter experiments are carried out. to know with certainty what and where is the rate-limiting This point is illustrated in Figure 2. Figure 2B shows step for the measured uptake of radiolabel. This question an idealized time course for the uptake of a solute (de- is of central importance in flux studies with any cell type, noted by S) that equilibrates rapidly between the erythro- but it is of particular concern in malaria-infected erythro- cyte cytosol compartment and the extracellular medium cytes, for reasons relating to both the complex compart- (Phase I) and is then either sequestered into the parasite, mentalization and active metabolism of the parasitized metabolized (to an impermeant form, denoted by S⬘), or cell. both, at a much slower rate (Phase II). Under these con- The conventional assumption in interpreting influx ditions, uptake of radiolabel will provide a true measure data derived from intact malaria-infected erythrocytes is of the transport of the solute across the RBCM only if it is that the first membrane encountered by a solute added to measured over the very early portion of Phase I of the the extracellular solution is the RBCM, and that the trans- time course. The use of longer time periods that fall port across this membrane, into the erythrocyte cytosol, outside this initial linear phase will lead to an underesti- therefore provides the rate-limiting step for the initial mate of the transport rate, as well as an overestimate of phase of solute uptake. However, this assumption is chal- IC50 values for inhibitors and of Michaelis constant (Km) lenged by the suggestion that there may be pathways that values for saturable transport processes. allow extracellular solution to come into direct contact Figure 2C shows an idealized time course for solute with the surface of the intracellular parasite and/or that uptake under conditions in which the initial transport step allow extracellular solutes to enter the parasite without (Phase I) is significantly slower than the subsequent actually entering the erythrocyte cytosol (see sect. IVB). If step(s) (Phase II), so that in practice, no sooner has a such pathways do exist, then the uptake of labeled solute solute entered the host cell cytosol then it finds itself 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 equili- bration step (Phase I) is significantly slower than the sub- sequent step(s) (Phase II) so that the solute is sequestered and/or metabolized immediately on entering the parasit- ized cell. The “distribution ratio” is the total concentration of the solute (S ⫹ S⬘) inside the cell, relative to that in the extracellular solution. Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 501 sequestered and/or metabolized. Under these conditions, side the initial part of the time course, then the amount of the uptake of radiolabel may be rate-limited by the trans- radiolabel taken up will be affected by both the rate of port of solute across the RBCM for an extended period, transport and by the subsequent conversion or compart- during which the total concentration of radiolabel inside mentalization rate. In this case, the characteristics that the cell may reach a much higher level than in the extra- emerge from such an analysis (kinetic constants, pharma- cellular solution. This does offer significant advantages to cological properties) may be a combination of those of the experimenter who, apart from anything else, will be the transport step and those of the intracellular pro- able to use less radiolabeled substrate to make a quanti- cess(es). tative estimate of the influx rate. However, it also holds significant dangers. First, as discussed in general terms by Wohlhueter C. Isosmotic Hemolysis and Plagemann (349), if the concentration of metabolized or sequestered radiolabel is very large relative to the An alternative method that has been used extensively concentration of unaltered solute in the erythrocyte cy- to study the altered permeability of the malaria-infected tosol, then the uptake time course might appear to ex- erythrocyte, as well as various other induced-permeability trapolate through the origin, while not truly doing so (i.e., phenomena in erythrocytes, involves suspending the cells the time course may appear to take the form of Fig. 2C, in an isosmotic solution of the solute of interest. The whereas the real situation is actually that of Fig. 2B). This principle behind this method is illustrated in Figure 3. On leads to an underestimate of the influx rate. suspension of the cell in the isosmotic solution, there is a Second, even if under one set of conditions the rate large inward concentration gradient, and hence a large of metabolism or sequestration (i.e., Phase II) is truly driving force for the influx of the extracellular solute much greater than the rate of influx (Phase I), so that the (represented by solid circles). If the permeability of the rate of accumulation of radiolabel provides an accurate RBCM to this compound is higher than that to the solutes measure of the initial transport rate, this will not neces- comprising the cell cytosol (represented by open circles), sarily be the case under all conditions. If, in investigating the rate of influx of material into the cell exceeds the rate the effects of different experimental conditions (e.g., in- of efflux, resulting in a net uptake of solute and water. creasing substrate concentration, addition of competitive This causes cell swelling and eventual hemolysis, the rate substrates or of potential inhibitors), a particular maneu- of which provides a semi-quantitative estimate of the ver reduces the rate of the metabolic or intracellular (net) rate of influx of solute. Hemolysis is readily moni- compartmentalization step (Phase II) while having a tored by measuring the release of hemoglobin (spectro- lesser effect on the initial transport step (Phase I), there is photometrically, using absorbance at 540 nm), or that of a risk that the compartmentalization process will become other intracellular solutes (e.g., ATP; Refs. 40, 166). the rate-limiting process. In this case, the situation will The isosmotic hemolysis technique has been used to revert to that represented in Figure 2B. If, under these investigate the permeability of the malaria-infected eryth- conditions, the length of the uptake incubation falls out- rocyte to a wide range of nonelectrolytes (127, 128, 179) 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 (F) at a rate greater than the efflux of cytosolic solutes (E) gives rise to a net uptake of solute and water, leading ultimately to hemolysis. Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 502 KIARAN KIRK Volume 81 and to a number of cations (178, 179, 303). It may be 5) The technique is of limited use in comparing the adapted for use with anions, although this requires that permeation of different solutes (or the effects of inhibi- the permeability of the cell membrane to cations be tors on the influx of different solutes) as the different higher than its permeability to the anions of interest. It is isosmotic solutions provide quite different extracellular only under this condition that the net influx of the anion environments and the properties (e.g., inhibitor sensitiv- of interest is limited by the permeability of the anion ity; see Ref. 179) of the pathways of interest may well vary itself, and not by the permeability of the accompanying between these different conditions. cation (as would normally be the case). In practice, this In summary, the isosmotic hemolysis technique pro- can be achieved by the use of NH⫹ 4 salts of the anions of vides a semi-quantitative measure of net solute perme- interest (128). NH⫹ 4 is not itself highly permeant but is in ation rates under limited (nonphysiological) conditions. It rapid equilibrium with NH3, which traverses the mem- offers a convenient means for testing relative potencies of brane rapidly, thereby providing an effective means for different inhibitors on the transport of any given substrate NH⫹ 4 to enter the cell (197). (albeit under extremely nonphysiological conditions). The isosmotic hemolysis technique offers the major However, as noted in point 5 (above), caution must be advantages of requiring relatively small amounts of mate- exercised in using this approach to compare the effect of rial (the spectrophotometric determination of hemoglo- one or more given inhibitors on the transport of different bin concentration is highly sensitive and allows the de- substrates. tection 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 D. Fluorescence solutions of many of the solutes of interest and therefore do not contribute to measured hemoglobin release) and of 1. Fluorescent transport solutes not requiring the use of expensive radioisotopes. Cabantchik, Ginsburg, and colleagues have used both However, it also has the following significant limi- the efflux (193, 195) and influx (34) of the fluorescent tations. anion NBD-taurine to probe the altered permeability 1) Its application is restricted to solutes that are properties of the parasitized erythrocyte. In the efflux sufficiently hydrophilic to be soluble at the concentra- experiments, cells were preloaded with the fluorescent tions needed to make an isosmotic solution (i.e., ⬃300 solute, washed, then suspended at low hematocrit in sa- mM for nonelectrolytes and ⬃150 mM for monovalent line. The fluorescence of the suspension increased as the salts) and which are not hemolytic to normal erythrocytes compound effluxed from the cells. This approach offers at these high concentrations. an advantage over analogous radiotracer experiments in 2) It requires that the cells be exposed to conditions allowing “on-line” measurements. However, it is re- that are far from physiological. This may affect the oper- stricted to fluorescent (and hence relatively large non- ation of the pathways of interest. physiological) substrates. It is also difficult to know with 3) The technique provides information about the net certainty which membrane in the infected cell constitutes influx of a particular solute under conditions in which the the rate-limiting step for the efflux of the fluorescent cell is exposed to a single, high concentration of that probe that remains in the cell after the initial wash pro- solute. If the influx pathway is saturated by high concen- cedure. trations of the solute of interest, the rate of hemolysis will More recently, larger fluorescent molecules such as not be indicative of the true permeability of the pathway Lucifer yellow (141, 199) and various fluorescent macro- to the solute. molecule conjugates (138, 153, 253) have been used in 4) The rate of hemolysis is influenced not only by the conjunction with fluorescence microscopy to study the net influx rate of extracellular solute but by the fate of the uptake of such solutes into individual parasitized eryth- solute once it has entered the infected cell. In Figure 3, rocytes. The data are qualitative and, as discussed in the solute is shown as being excluded from the intracel- section IVC, may, in some cases, be compromised by the lular parasite and remaining unaltered and in free solution dissociation of the fluorophore from the molecules of within the erythrocyte cytosol. However, if the solute interest (153, 291). enters the parasite and is either metabolized or bound, in such a way as to change its osmotic contribution, then the 2. Fluorescent ion indicators amount of solute that will have to enter the cell to pro- duce a given amount of cell swelling (and, ultimately, Over the last decade, the study of ion transport in hemolysis) may be either more or less than if this does not animal and plant cells has been revolutionized by the use occur. Under such circumstances, estimates of the rela- of ion-sensitive fluorescent indicators that can be loaded tive permeation rates of different solutes from relative into cells and thereby used to monitor the intracellular rates of hemolysis are, at best, semi-quantitative. concentrations of a range of different ions. Ions for which Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 503 fluorescent indicators are available include H⫹, Na⫹, K⫹, by Lee et al. (200) who used X-ray microanalysis in Ca2⫹, Mg2⫹, and Cl⫺. Those for H⫹ and Ca2⫹ are in conjunction with electron microscopy to obtain esti- routine use in a wide range of cell types and have pro- mates of the Na⫹, K⫹, Cl⫺, and phosphorous content of vided a wealth of information on the regulation of these the different compartments of the malaria-infected two ions. The use of indicators for the other ions is less erythrocyte. The transport of monovalent inorganic cat- straightforward and has been much more limited. ions in the parasitized erythrocyte is discussed in detail Although fluorescent ion indicators have not, as yet, in section IX J. been widely applied to the study of the intracellular ma- laria parasite, there have been a number of recent studies demonstrating the applicability of this approach. F. Electrophysiological Techniques Mikkelsen et al. (225) used the pH-sensitive fluorescent dye 2⬘,7⬘-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) The patch-clamp technique involves the formation to measure the intracellular pH (pH i ) of parasites of a high-resistance (giga-ohm) seal between a cell (P. chabaudi) freed from their host cells using N2 cavita- membrane and a glass micropipette, then monitoring tion (see sect. IIIA2D), whereas Bosia et al. (27) used the currents arising from the flux of ions either across 6-carboxyfluoroescein to measure the pHi of parasites the enclosed patch of membrane or across the whole (P. falciparum) within erythrocytes permeabilized using cell membrane (see Ref. 155). This technique has Sendai virus (see sect. IIIA2B). More recently, Wunsch and proven invaluable in elucidating the characteristics of colleagues have described the use of BCECF in conjunc- tion with a digital imaging system, to monitor the cytoso- ion channels in many animal and plant cells, but it has lic pH of the parasite [both within intact erythrocytes and not, as yet, been widely applied to parasitic protozoa. in parasites released from their host erythrocytes using Patch-clamping malaria-infected erythrocytes is not the peptide hemolysis technique described in sect. IIIA2C; straightforward. The infected cells are, compared with (355, 356)] and the pH in the cytoplasm of the host eryth- the cell types with which most electrophysiologists are rocyte (see sect. IX J). The same group has used the familiar, both small and fragile, with a tendency to either Na⫹-sensitive dye benzofuran isophthalate acetoxymethyl burst or to disappear up into the patch pipette on appli- ester (SBFI) to monitor the concentration of Na⫹ within cation of suction. The earliest mention in the literature of the intracellular parasite (see sect. IX J) (354, 356). patch-clamp data from intact, malaria-infected erythro- Several other groups have reported the use of the cytes of which I am aware is in a review by Cabantchik fluorescent Ca2⫹ indicators indo 1, fluo 3 (1), and fura 2 (39) which refers to unpublished data (from Stutzin and (102) to estimate cytosolic Ca2⫹ concentrations in intact Cabantchik) suggesting the presence of a voltage-depen- and/or permeabilized malaria-infected erythrocytes, as dent, phloridzin-sensitive ion channel in the infected cell well as the use of the colourimetric Ca2⫹ indicator ar- membrane. However, the data were not presented. senazo III (242), in isolated parasites. The transport and In a study of Ca2⫹ transport in the malaria-infected homeostasis of Ca2⫹ in the malaria-infected erythrocyte is erythrocyte, Desai et al. (64) reported a series of cell- discussed in detail in section IXK. attached patch-clamp measurements on intact parasitized erythrocytes. In these experiments they observed (in 2 of E. Ion Analysis 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. Early estimates of the Na⫹/K⫹ composition of ma- IXK). Very recently, Desai and colleagues (62a, 67) have laria-infected erythrocytes were made using flame pho- reported obtaining both whole cell and cell-attached re- tometry of extracts of erythrocytes from malaria-in- cordings of intact, trophozoite-stage parasitized erythro- fected animals (74). These measurements did indicate a perturbation of the normal Na⫹/K⫹ balance in infected cytes and obtained evidence for a novel, voltage-depen- erythrocytes; however, the conclusions that could be dent anion channel (see sect. VC3). drawn were limited by the multi-compartmental nature Desai et al. (63) have also described single-channel of the parasitized cell. Ginsburg et al. (124) used flame recordings from the PVM enclosing parasites freed from photometry, in combination with Sendai virus perme- their host erythrocytes using two different techniques abilization of the host cell membrane (see sect. IIIA2B), (digitonin and an electrical pulse applied to the host cell to estimate the Na⫹/K⫹ concentration ratio in the host membrane). Similar recordings were obtained in a study cell and parasite compartments of malaria-infected in which the membrane fraction of homogenized intact cells, showing it to be increased to well above normal parasitized erythrocytes were reconstituted into a planar levels in the red cell cytosol but maintained at a low lipid bilayer (65). The characteristics of this channel are level within the parasite. Similar results were obtained discussed in section VIB. Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 504 KIARAN KIRK Volume 81 G. Genetic Techniques presence in these cells of an array of endogenous trans- porters and channels, some of which are activated in The techniques of modern molecular biology have, response to the expression of “foreign” proteins (e.g., over the past decade, yielded sequences of a number of Refs. 38, 292, 326). putative plasmodial membrane transport proteins. In all The ability of at least one plasmodial ABC protein to cases, this has involved cloning homologs of transporters complement a transport-deficient yeast strain (340) indi- from other organisms. These include a number of P-type cates that yeast might be a suitable system in which to ATPases (75, 172, 188, 189, 324, 325), two V-type ATPase clone (by complementation) and/or characterize plasmo- subunits (170, 171), several members of the ABC trans- dial transporters and channels. This approach has proven porter family (29, 96, 347), and homologs of the mitochon- highly successful in the identification and characteriza- drial ATP/ADP exchanger (76, 149, 150) and phosphate tion of a range of transporters and channels from plants transporter (20). (98) but has not, as yet, been widely used in other or- The malaria genome sequencing project is now ganisms. nearing completion (58, 104, 306). The recently pub- Other approaches still in their infancy in this field but lished sequence of chromosomes 2 and 3 of P. falcipa- which will, in the longer term, yield vital information rum include a total of seven putative transporter se- regarding the function and physiological role(s) of the quences (28, 104) and, as the genome sequencing proteins of interest within the parasite include the use of project progresses, a wealth of other such sequences antisense oligonucleotides (14, 15, 59, 257), ribozymes are becoming available. This poses a major challenge to (93), gene knockout (53, 344), and gene transfection (334, those in the field. Functional expression of malaria- 344, 352). encoded membrane proteins is difficult, particularly if they are large (as is likely to be the case for many IV. SOLUTE TRAFFICKING ROUTES IN THE transporters and channels). The recent reports of in- PARASITIZED CELL creased transport of several solutes into Xenopus oo- cytes injected with P. falciparum mRNA (247) and the successful expression of cloned P. falciparum hexose A. Windows, Tubes, Vesicles, and Ducts (190, 190a, 349a, 350) and nucleoside (44, 241b) trans- porters indicate that the Xenopus oocyte is likely to be According to the traditional view of the malaria-in- an extremely useful tool for the characterization of fected erythrocyte, represented in Figure 4A, the move- plasmodial transport proteins, as well as, perhaps, for ment of solutes between the intracellular parasite and the the identification of novel transport proteins by expres- external milieu occurs via the erythrocyte cytoplasm. Sol- sion cloning (247). However, the Xenopus oocyte sys- utes taken up into the intracellular parasite have first to tem does have limitations, not least of which is the gain entry to the erythrocyte, across the RBCM. From 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 extracel- lular 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). Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 505 here they can move into the parasite either by being B. Does the Intracellular Parasite Have Direct transported sequentially across the PVM and PPM or by Access to the Extracellular Medium? endocytosis (see sect. VII). In recent years there has been considerable interest A number of experimental observations have been in the possibility that there is, in addition to the “sequen- interpreted in terms of the existence of a mechanism that tial route” (in which solutes cross each of the three mem- allows solutes to pass between the intracellular parasite branes in sequence) outlined above, one or more addi- and the external medium, without entering the host cell tional “parallel routes” that allow solutes to move cytosol. between the parasite and the external medium, without In two intriguing studies, Cabantchik and colleagues their actually entering the erythrocyte cytosol. There is (209) showed that two different Fe3⫹ chelators (desferri- evidence that the plasma membrane of the parasitized oxamine and a fluorescent derivative thereof) and the erythrocyte is incapable of endocytosis (143, 251), as is bioflavonoid glycoside phloridzin (208) were toxic to the thought to be the case for the normal, mature erythrocyte. parasite when added to the extracellular solution, but had It remains controversial, however, whether there might little effect on the parasite when they were encapsulated be some means by which the parasite accesses the exter- (at much higher concentrations) within red blood cells nal medium other than via transport across the host eryth- that were subsequently infected by the parasite. In inter- rocyte membrane, into the red cell cytoplasm. preting these results, the authors proposed that these As long ago as 1973, Bodammer and Bahr (24) pro- reagents cannot enter the parasite from the red cell cy- posed, on the basis of scanning and transmission electron tosol but are able to do so only from the external solution micrographs of P. berghei-infected mouse erythro- (via some form of parallel route). This interpretation is cytes, that a localized region of apposition of the intracel- consistent with the data; however, alternative explana- lular parasite to the red blood cell surface might serve as tions cannot be ruled out. “a specialized entry and exit site for metabolites” and One possibility is that one or more of the agents coined the phrase metabolic window (Fig. 4Ba). Lauer et tested exert their cytotoxic effects at the external surface al. (199) have recently proposed a variation of this model of the infected cell, perhaps by blocking the uptake of in which specialized regions of membrane formed at nutrients and/or the release of metabolic wastes (80). points of contact between the TVM and the RBCM serve Phloridzin does block the induced transport of small sol- utes into parasitized cells (194, 293). However, the same is as a route of entry for low-molecular-weight solutes into not known to be true of the Fe3⫹ chelators, and there is the TVM network, from where they are taken up by the evidence that desferrioxamine exerts its antiplasmodial parasite (Fig. 4Bb). However, much of the recent atten- effect from within the parasite (283). tion has focused on the proposal from Taraschi and col- Another possibility is that in the experiments with leagues (253) that the parasite has direct access to the cells preloaded with the different antiplasmodial agents extracellular solution via a so-called “parasitophorous then invaded by the parasite, leakage of the compounds duct,” a tubular membranous structure that extends be- from the cytosol of the infected erythocytes into the tween the parasitophorous vacuole membrane and the extracellular medium reduced their concentration (both erythrocyte membrane. The duct, as originally proposed, inside and outside the cell) to below that required to exert would allow the parasite plasma membrane to come into an antiplasmodial effect. Parasitized erythrocytes do have direct contact with the extracellular solution (Fig. 4C) a substantially increased permeability to a wide range of and would provide a means for the intracellular parasite solutes (sect. V), and Loyevsky and Cabantchik (208) dem- to take up macromolecules from the external medium, onstrated that erythrocytes preloaded with the different across the PPM, by a process of endocytosis. This pro- reagents did lose the majority to the external medium, posal has been the subject of considerable controversy particularly once the parasites reached the mature tro- and in the heated debate surrounding the question of phozoite-schizont stage (which is when the different whether the duct exists, there has been a tendency for a drugs of interest exert their major antiplasmodial effect). number of related but separate issues to become inter- It was argued that the concentration remaining within the twined. Here, two issues are considered separately. The infected cell should have been more than enough to retard first is the question of whether there is some form of parasite growth. However, it was not demonstrated that parallel route that allows solutes to move between the the drug retained by trophozoite-infected cells was actu- intracellular parasite and the external solution, without ally in the erythrocyte cytosol. At least some may have actually entering the erythrocyte cytosol. The second is been taken up into the parasite’s food vacuole in the the question of whether the malaria-infected erythrocyte endocytotic feeding process (138, 319), before the induc- has the capacity to take up at least some macromolecules tion of NPP in the RBCM and before the parasites become from the extracellular medium. sensitive to the drug. Once there it may have been Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 506 KIARAN KIRK Volume 81 trapped, unable to gain access to potential targets else- C. Does the Parasitized Erythrocyte Take Up where in the parasite. Macromolecules and Other A separate line of evidence for the existence of par- High-Molecular-Weight Solutes? allel routes comes from confocal microscopy studies of parasitized erythrocytes incubated with various fluores- The existence of tubular structures traversing the cent solutes, including several fluorescently labeled mac- cytosol of malaria-infected erythrocytes was described by romolecules and the smaller, widely used endocytosis Grellier et al. (139). However, it was Taraschi and col- marker Lucifer yellow. Papers describing a number of leagues (138, 253, 313) who first proposed that these tubes such studies report that fluorescence was localized to the mediate the trafficking of macromolecules with diameters intracellular parasite, and associated tubular structures in of up to 50 –70 nm between the external medium and the the host cell compartment, while apparently remaining parasite, and who coined the term parasitophorous duct (see Fig. 4C). This hypothesis was first proposed on the excluded from the bulk host cell cytosol (138, 199, 252, basis of experiments in which it was shown using confo- 253). In the case of the fluorescently labeled macromole- cal microscopy that macromolecules (e.g., fluorescent cules, concerns have been raised about dissociation of the dextrans, biotinylated protein A, IgG antibody) and fluo- fluorescent label (see sect. IVC). However, this issue not- rescent latex beads, added to the extracellular medium, withstanding, the question still arises of why in such gained access to the aqueous space surrounding the par- experiments the fluorescence appears in the parasite and asite. In cells incubated with the fluorescent beads, fluo- associated tubular structures, but not in the erythrocyte rescence was shown to be associated with tubular struc- cytosol. The data have been interpreted as indicating that tures that were proposed to connect the parasitophorous the fluorescent solutes are taken up directly into the vacuole and host erythrocyte membranes. parasite from the external medium (138, 252, 253). There The experiments of Pouvelle et al. (253) have been are, however, a number of technical considerations, some questioned on a number of technical grounds. Fujioka and or all of which may be relevant. Aikawa (99, 100) demonstrated that parasitized erythro- The composition of the red cell cytosol is quite dif- cytes that had been maltreated in various ways took up ferent from that of the interior of the parasite and the colloidal gold and fluorescent dextrans, whereas parasit- TVM system, and it is possible that there is significant ized cells maintained under normal conditions did not. interference by components of the erythrocyte cytosol (in This prompted the suggestion that the uptake of macro- particular the hemoglobin) with fluorescent signal arising molecules described by Pouvelle et al. (253) was due to from this compartment. It is also possible that the fluo- the parasitized erythrocytes used in this earlier study rescent compounds are somehow accumulated within the having been exposed to adverse conditions (99, 100), a parasite and the compartment(s) enclosed by the TVM, to contention strongly rejected by Taraschi and Pouvelle levels substantially higher than those reached in the eryth- (313, 314). rocyte cytosol. Both situations would tend to give the Several others have emphasized potential problems appearance of there being negligible fluorescent com- arising from the dissociation of low-molecular-weight flu- pound in the host cell compartment, while not actually orophores from the fluorescently labeled probes used in being the case. the original study (143, 153, 291). In particular, Hibbs et al. Another possibility is that the lack of fluorescence (153), using a combination of confocal and electron mi- croscopy, demonstrated that although incubation of ma- associated with the host cell cytosol is due simply to the laria-infected erythrocytes with the fluorescent beads compounds leaking out of this compartment before (and used in the original study by Taraschi and colleagues perhaps during) the confocal microscopy measurements. resulted in fluorescent labeling of the parasite and, in In the majority of experiments of this sort, parasitized some cases, of associated tubular structures, the beads erythrocytes were preincubated for prolonged periods themselves (which had diameters down to 14 nm, well (typically 30 –120 min) in the presence of fluorescent sol- below that of the putative duct) remained excluded from ute, then the “loading solution” was removed by washing the parasitized erythrocyte. The labeling of the parasite in the cells repeatedly before confocal measurements were this study was attributed to the release of membrane- made. It is conceivable that during the wash procedure, permeant fluorescent dye from the beads during the in- and subsequently, the fluorescent compound was lost cubation period, and it was suggested that the same phe- from the host cell compartment, perhaps via NPP induced nomenon was responsible for the original results reported by the parasite in the host cell membrane (see sect. VC). by Pouvelle et al. (253). In summary, although there are several independent Using thin-layer chromatography, Goodyer et al. lines of evidence in support of the existence of parallel (138) demonstrated that the fluorescent dextrans used in routes in the malaria-infected erythrocyte, none is entirely the initial work of Pouvelle et al. (253) did undergo sig- conclusive, and the issue awaits further clarification. nificant degradation during a 4-h incubation period. How- Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 507 ever, ⬍0.0001% of the fluorophore molecules were re- other explanations (e.g., uptake of radiolabeled products leased. It was argued that this could not account for the of oligonucleotide degradation) might account for the observed uptake of fluorescence by parasitized erythro- results described. cytes; however, it was not actually demonstrated that the In a number of the studies of the antiplasmodial fluorescence taken up into the intracellular parasite was effect of antisense oligonucleotides, parasite growth was in the form of the macromolecular dextran conjugate, and measured using asynchronous cultures and/or measured the data presented do not exclude the possibility that the over a period that encompassed one or more schizogony fluorescence associated with the parasite is in the form of and reinvasion steps. The data from these papers do not low-molecular-weight fluorophore molecules taken up exclude the possibilities that the reagent(s): 1) targeted from the external medium and perhaps concentrated the merozoites during the brief period in between their from the extremely low levels in the extracellular solution release from one cell and invasion of another, 2) inhibited to relatively high levels within the intracellular parasite. parasite invasion, or 3) entered the parasitophorous vac- Goodyer et al. (138) have also presented electron uole in sufficient quantity during the endocytotic invasion microscopic evidence for the uptake of ruthenium red, an process to cause the subsequent retardation of parasite electron-dense marker into ductlike structures that ap- growth. However, in at least one study, oligonucleotides peared to interconnect the erythrocyte membrane and the (ribozymes) were shown to exert a significant sequence- PVM. These findings would appear to be directly at odds specific antiplasmodial effect within a single intraerythro- with those of Elford and colleagues (80, 89), who have cytic cycle (measured over 24 h after their addition to presented evidence that in parasitized erythrocytes ex- early ring-stage parasites; Ref. 93). In this case at least, posed to ruthenium red, the compound remains entirely there is therefore reason to believe that the oligonucleo- excluded from the infected cell. This is difficult to recon- tides entered the parasitized erythrocyte at some time cile with the existence of a duct, as is the earlier finding subsequent to the initial invasion step. by a number of groups (including that of Taraschi and Oligonucleotides are not the only high-molecular- colleagues) that parasitized erythrocytes fail to take up weight solutes reported to inhibit the growth of the intra- fluorescent molecules that have dimensions well below cellular malaria parasite. Gelonin, a single peptide chain the diameter of the proposed duct (143, 251). protein inhibitor of protein synthesis, has been shown to In addition to the various papers claiming to demon- inhibit parasite proliferation when exposed to parasitized strate directly the uptake of high-molecular-weight sol- erythrocytes for a fixed period within a single erythrocytic utes into the malaria-infected erythrocyte (138, 252, 253), cycle (235). Dermaseptins, linear polycationic peptides there are a number of studies that have been cited as composed of 28 –34 amino acids, have also been shown to providing independent evidence for the uptake by para- gain access to the intracellular malaria parasite within sitized erythrocytes of at least some such solutes. These include a number of demonstrations that antisense oli- seconds of their addition to P. falciparum-infected hu- godeoxynucleotides and ribozymes (i.e., oligonucleotides man erythrocytes and to inhibit the growth of the parasite incorporating a sequence able to mediate the cleavage of (114). The dermaseptins are amphipathic and do interact complementary mRNA), targeted against parasite-en- with lipid bilayers. Although it was argued that they do coded enzymes, inhibit the growth of the malaria parasite. not translocate across the plasma membrane of normal Following on from the original reports of antisense oligo- uninfected erythrocytes, the data do not exclude the pos- nucleotides inhibiting parasite proliferation (59, 257), it sibility that these compounds enter parasitized cells via was suggested that this was a nonspecific effect arising the lipid phase of the RBCM. from the polyanionic oligonucleotides interfering with the Very recently it has been reported that addition to the invasion of the erythrocyte by the parasite (49, 256). It culture medium of a 93-amino acid fragment of the en- was shown subsequently, however, that although these zyme ␦-aminolevulinate dehydratase inhibits parasite reagents show sequence-independent effects when used growth (25a). It was shown using both immunofluores- at concentrations ⬎1 ␮M, at lower concentrations their cence and a radiolabeled form of the polypeptide that the effects are sequence specific (14, 15). The same has also molecule (termed ALAD-⌬NC) was taken up by infected been shown to be true of ribozymes (93). The conclusion but not uninfected cells. The radiolabel experiments pro- to be drawn from this work is that the oligonucleo- vided evidence that the polypeptide was present within tides are somehow gaining access to the interior of the the parasite (including the food vacuole) but not in the parasite. erythrocyte cytosol, although the mechanism of uptake In the original paper describing the antiplasmodial was not investigated. activity of antisense oligonucleotides, it was reported that There have also been reports that antibodies directed radiolabeled antisense oligonucleotides were taken up by against antigens localized within the parasitized erythro- infected, but not normal, erythrocytes (257). However, cyte inhibit parasite growth (169). However, the mecha- the data were not presented, and it is not clear whether nism by which they do so is unclear, and it has not been Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. 508 KIARAN KIRK Volume 81 TABLE 1. High-molecular-weight solutes for which cellular medium and the intracellular parasite without evidence has been presented for and/or actually entering the erythrocyte cytosol, and 2) whether against their being taken up by Plasmodium there is a mechanism by which the parasite is able to take falciparum-infected erythrocytes up macromolecules and other high-molecular-weight sol- Solute Evidence Reference Nos. utes from the extracellular solution, have been, and re- main, contentious. There is substantial evidence against Taken up the existence of a parasitophorous duct in the form orig- Oligonucleotides inally proposed (253). Nevertheless, there is sufficient Antisense RNA Sequence-specific 15 evidence in support of both hypotheses to warrant further inhibition of investigation. parasite growth Ribozymes Sequence-specific 93 inhibition of parasite growth V. THE RED BLOOD CELL MEMBRANE Oligopeptides/proteins Antibodies Inhibition of parasite 169 A. General Considerations growth Quenching of 253 fluorescent label at The mature human erythrocyte membrane is en- the PPM dowed with a plethora of membrane transport systems. Rhodamine-protein A Fluorescence 253 micrographs In at least some cases these serve no known purpose in Gelonin Inhibition of parasite 235 the mature erythrocyte but are thought to be the ves- growth tiges of those required for the much higher metabolic Dermaseptins Inhibition of parasite 114 growth/fluorescence and biosynthetic requirements of the cells from which micrographs the erythrocyte is derived. Many of these endogenous ALAD-⌬NC Inhibition of parasite 25a transport pathways have been characterized at a func- growth/fluoresence micrographs/uptake tional level, with their kinetic and pharmacological of radiolabel properties described in detail. Some have been identi- Other fied at a molecular level and are homologous to trans- Ruthenium red Electron micrographs 138 port proteins in other tissues. Fluorescently labeled Fluorescence 253 For some solutes there are a number of alternative dextrans micrographs Fluorescently labeled Fluorescence 253 transport pathways across the erythrocyte membrane, all latex beads micrographs of which may contribute to the measured influx or efflux. For example, the erythrocyte has at least four discrete Not taken up and well-characterized K⫹ transport mechanisms (the Ruthenium red Electron micrographs 89 Na⫹/K⫹ pump, the NaKCl2 cotransporter, the KCl cotrans- Fluorescently labeled Fluorescence 143 porter, and the Ca2⫹-activated K⫹ channel) as well as dextrans micrographs Fluorescently labeled Fluorescence 153 others that are less well understood (e.g., Ref. 19). Amino latex beads micrographs/electron acids are transported across the erythrocyte membrane micrographs via a number of different systems with overlapping spec- PPM, parasite plasma membrane. ificity, e.g., at least five different pathways contribute to the flux of glycine across the erythrocyte membrane un- der physiological conditions (86). Similarly, the monova- demonstrated that these antibodies are actually taken up lent anion lactate permeates the membrane via at least into intact parasitized cells. three distinct pathways: a monocarboxylate carrier, the Table 1 provides a summary of the results of those band 3 anion exchanger, and simple diffusion of the pro- studies that provide evidence in support of the view that tonated acid across the bilayer (68, 250). In many cases, the malaria-infected erythrocyte is able to take up mac- these alternative pathways can be distinguished on the romolecules and other high-molecular-weight solutes basis of their different pharmacological and kinetic prop- from the external medium, as well as listing those which erties. would argue against there being a nonspecific uptake of For any perturbation that causes an increase in the such solutes. rate of transport across the erythrocyte membrane, the question arises of whether the increase is due to a change D. Summary in the activity of endogenous systems or to the induction of new pathways. In the case of malaria infection (in The two related questions of 1) whether there is a which the parasite invades only a fraction of the erythro- mechanism by which solutes can pass between the extra- cytes available to it either in the bloodstream or in cul- Downloaded from journals.physiology.org/journal/physrev at UPM Montpellier Biblio (162.038.196.201) on November 13, 2024. April 2001 MEMBRANE TRANSPORT IN THE MALARIA-INFECTED ERYTHROCYTE 509 ture), the further question arises of whether an apparent the saturable component of tryptophan influx. The most increase in the flux via an endogenous pathway is due to striking examples of this phenomenon, however, come a genuine change in the activity of that pathway in para- from experiments with erythrocytes taken from malaria- sitized cells, or to the parasite invading preferentially a infected animals. subpopulation of cells that have transport activity differ- Parasitized erythrocytes from monkeys infected with ent from that of the population as a whole. Reticulocytes P. knowlesi (4) and from mice infected with P. vinckei and young erythrocytes have higher activity of many vinckei (302) both show increased uptake of choline via a transport systems than do mature erythrocytes (e.g., Refs. pathway that has the same Michaelis constant (Km) and 145, 182). Thus, if the parasites have a significant prefer- pharmacological characteristics as the endogenous cho- ence for younger over older cells, th