Plasmodium falciparum-activated Chloride Channels Are Defective in Erythrocytes from Cystic Fibrosis Patients

Abstract

An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the “new permeation pathway” for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF. An inwardly rectifying anion channel in malaria-infected red blood cells has been proposed to function as the “new permeation pathway” for parasite nutrient acquisition. As the channel shares several properties with the cystic fibrosis transmembrane conductance regulator (CFTR), we tested their interrelationship by whole-cell current measurements in Plasmodium falciparum-infected and uninfected red blood cells from control and cystic fibrosis (CF) patients. A CFTR-like linear chloride conductance as well as a malaria parasite-induced and a shrinkage-activated endogenous inwardly rectifying chloride conductance with properties identical to the malaria-induced channel were all found to be defective in CF erythrocytes. Surprisingly, the absence of the inwardly rectifying chloride conductance in CF erythrocytes had no gross effect on in vitro parasite growth or new permeation pathway activity, supporting an argument against a close association between the Plasmodium-activated chloride channel and the new permeation pathway. The functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF. Intraerythrocytic development of malaria parasites leads to increased nutrient uptake through parasite-induced new permeation pathways (1Kirk K. Physiol. Rev. 2001; 81: 495-537Crossref PubMed Scopus (328) Google Scholar, 2Kirk K. Nature. 2000; 406: 949-951Crossref PubMed Scopus (11) Google Scholar, 3Ginsburg H. Stein W.D. Biosci. Rep. 1987; 7: 455-463Crossref PubMed Scopus (23) Google Scholar, 4Ginsburg H. Novartis Found. Symp. 1999; 226: 99-108PubMed Google Scholar) that are considered promising targets for antimalarial chemotherapy development (5Kirk K. Staines H.M. Martin R.E. Saliba K.J. Novartis Found. Symp. 1999; 226: 55-66PubMed Google Scholar). Previous reports demonstrated that the new permeation pathways show functional characteristics of a chloride channel (1Kirk K. Physiol. Rev. 2001; 81: 495-537Crossref PubMed Scopus (328) Google Scholar). More recent data suggested that a small conductance chloride channel, as measured with the on-cell patch clamp configuration, accounts for the increased uptake of nutrients into infected red blood cells and that thus this channel qualifies as a prime target for pharmacotherapy (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar). The channel exhibits voltage-dependent gating, showing higher open probability at negative membrane potential, which is visible in the whole-cell patch clamp configuration as a strong inwardly rectifying chloride current. The pharmacological profile of this channel shows a striking resemblance to that of the cystic fibrosis transmembrane conductance regulator (CFTR), 1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; RT, reverse transcriptase; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; I-V, current-voltage; PKA, protein kinase A; PKI, PKA inhibitor.1The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; RT, reverse transcriptase; DIDS, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid; I-V, current-voltage; PKA, protein kinase A; PKI, PKA inhibitor. a cAMP-regulated chloride channel that is mutated in patients with cystic fibrosis (CF) (7Anderson M.P. Rich D.P. Gregory R.J. Smith A.E. Welsh M.J. Science. 1991; 251: 679-682Crossref PubMed Scopus (430) Google Scholar, 8Bear C.E. Li C.H. Kartner N. Bridges R.J. Jensen T.J. Ramjeesingh M. Riordan J.R. Cell. 1992; 68: 809-818Abstract Full Text PDF PubMed Scopus (778) Google Scholar). Functional expression of CFTR in red blood cells is suggested also by the failure of CF erythrocytes to perform deformation-induced and cAMP-induced ATP release (9Sprague R.S. Ellsworth M.L. Stephenson A.H. Lonigro A.J. Am. J. Physiol. 2001; 281: C1158-C1164Crossref PubMed Google Scholar, 10Sprague R.S. Ellsworth M.L. Stephenson A.H. Kleinhenz M.E. Lonigro A.J. Am. J. Physiol. 1998; 275: H1726-H1732PubMed Google Scholar). In the present study, direct evidence in support of this concept was obtained by patch clamp measurements of whole-cell chloride currents in CF and non-CF red blood cells. The CF defect in endogenous and parasite-induced chloride conductances was exploited to evaluate the possible contribution of red blood cell chloride channels to the new permeation pathway. Moreover, the lack of functional chloride channels in CF red blood cells offered us the unique possibility to study the putative obligatory role of the Plasmodium-activated chloride current in the growth and survival of Plasmodium falciparum. RT-PCR—For RT-PCR, RNA was isolated from human erythroblasts obtained by bone marrow puncture (11Von Lindern M. Zauner W. Mellitzer G. Steinlein P. Fritsch G. Huber K. Löwenberg B. Beug H. Blood. 1999; 94: 550-559Crossref PubMed Google Scholar) using TRIzol (Invitrogen). cDNA was prepared from 2 μg of total RNA using SuperScript II reverse transcriptase (Invitrogen). For amplification of CFTR, PCR reactions were performed using platinum Taq polymerase (Invitrogen) with primers 5′-AGGGGAAGTCACCAAAGCAGTACAGC-3′ (sense; position 402–427 in sequence NM_000492 (GenBank™/EBI accession number); exon 4) and 5′-GCGCAGAACAATGCAGAATGAGATG-3′ (antisense; position 1173–1149; exon 8). The thermocycler profile was as follows: 96 °C for 4 min followed by 11 cycles (1 min at 68 °C, 2 min at 68 °C, 1 min 45 s at 72 °C) with annealing at 68 °C, 11 cycles with annealing at 64 °C, 11 cycles with annealing at 60 °C, 17 cycles with annealing at 56 °C, and a final extension at 72 °C for 5 min. The high number of cycles was necessary due to very low amounts of CFTR mRNA. The amount of cDNA used in each reaction (1–2 μl) was normalized with respect to the amount of actin cDNA by performing PCR on serial dilutions of cDNA using primers specific for β-actin, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ (sense) and 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′ (antisense), under conditions similar to those used for the CFTR PCR but using a 25-cycle PCR program. Relative expression levels were compared with expression in T84 cells (which have high CFTR expression) by using dilutions (1000–100,000-fold) of cDNA from T84 cells. Although CFTR expression in erythroblasts was at least 1000-fold lower than in T84 cells, the product was detected in the majority of PCR reactions (n = 5–8 for each cDNA sample) and was always absent in a –RT control (n = 5), and the identity of the 771-bp PCR product was verified by sequencing using an ABI377 automated sequencer. Plasmodium Culture—P. falciparum strains FCR3 (Gambian origin) and NF54 (unknown origin) were cultured essentially by the method of Trager and Jensen (12Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6122) Google Scholar) in standard culture medium. Parasitemia was determined by evaluation of Giemsa-stained thin films. When needed, parasite cultures were synchronized using alanine treatment (13Braun-Breton C. Rosenberry T.L. da Silva L.P. Nature. 1988; 332: 457-459Crossref PubMed Scopus (129) Google Scholar). Invasion—Nycodenz-purified mature stages-infected red blood cells at 3% hematocrit were mixed with red blood cell suspensions of various origins at a ratio of 1:100. Triplicate 5-ml cultures of each red blood cell suspension were cultivated overnight to allow reinvasion. A sample of 150 μl from each of the cultures was then transferred to a 96-well plate, and 25 μl of [3H]hypoxanthine (ICN Biomedicals Inc., Irvine, CA) in standard culture medium (1 μCi) was added to each of the wells. Cultures were incubated for another 22.5 h and harvested using a Titertek cell harvester (ICN Biomedicals Inc.), and incorporation of radiolabel was determined by liquid scintillation counting using a Top-Count NXT (PerkinElmer Life Sciences) as a measure of parasite viability and invasion rate. The original 5-ml cultures were maintained for 7 days with periodic addition of fresh red blood cells and culture medium (identical treatment for all cultures), and parasitemia was regularly determined. Patch Clamp—Red blood cells were bathed in 110 mm CsCl, 5 mm MgSO4, 3.5 mm sodium gluconate, 12 mm HEPES, 8 mm Tris-HCl (pH 7.4), and 10 mm dextrose. In the shrinkage experiments the bathing solution contained an additional 300 mm mannitol. The intracellular pipette solution contained 110 mm CsCl, 2 mm MgSO4, 25 mm HEPES, 1 mm EGTA, 2 mm Na2ATP, 50 mm mannitol (pH 7.4). Patch pipettes were pulled from borosilicate glass (Clark GC150T-10) and had a resistance of 8–12 megohms. All experiments were done with 2 mm ATP in the pipette unless stated otherwise. Where stated, G449 (10 μg/ml IgG), CF-2 (0.1 mg/ml, preincubated with G449 for 5 min at room temperature), protein kinase A (PKA) inhibitor (PKI; 10 μm), or acid phosphatase (30 units/ml) was added to the pipette. Forskolin (1 μm), niflumic acid (100 μm), furosemide (100 μm), 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS; 250 μm), or glibenclamide (100 μm) was added to the bathing solution. Cell shrinkage was triggered by increasing the osmolarity of the bath by addition of 300 mm mannitol to the standard bathing solution following seal formation. In the figures, error bars indicate S.E., and the number of individuals tested is given between parentheses. Voltage steps from –120 to +120 mV were applied in 30-mV increments; holding potential was set at 0 mV. For command pulse control, data acquisition, and analysis, pCLAMP 6 software (Axon instruments Inc., Forester City, CA) was used. Isotonic Lysis—Synchronized infected cells (enriched to a parasitemia of 5–10%) from non-CF and CFTRΔPhe-508/ΔPhe-508 patients (n = 4 different persons) were bathed in an isotonic solution containing 300 mm glucose, 150 mm choline chloride, or 300 mm alanine. The cells were incubated at 37 °C for 15 min, and cell lysis due to the passive movement of the osmolyte into the cells was measured spectrophotometrically at 580 nm. Results are plotted as percentage compared with total lysis after 2 h of incubation in 300 mm choline chloride in the absence of inhibitors. Uninfected red blood cells did not undergo lysis in response to isosmotic choline or alanine. In case of glucose, the lytic response resulting from glucose uptake through the endogenous glucose carrier in uninfected red blood cells was subtracted. To characterize the role of the malaria-induced chloride channel activity in parasite blood stage development, we performed whole-cell patch clamp experiments with control red blood cells and red blood cells from CF patients, both uninfected and infected with P. falciparum. As a first step, to identify CFTR in red blood cells, we studied uninfected red blood cells from healthy individuals and homozygous ΔPhe-508 patients, the most common CFTR mutation in the Caucasian population. Red blood cells were freshly isolated from heparinized blood samples and were bathed at 37 °C in a 110 mm symmetrical CsCl solution. In the presence of ATP in the pipette solution, an average whole-cell chloride conductance of 873 ± 82 pS (n = 15 different individuals) was observed in red blood cells from healthy volunteers (Fig. 1A), which was virtually absent from CF red blood cells (131 ± 19 pS, n = 8 different CF (CFTRΔPhe-508/ΔPhe-508) patients) (Fig. 1B). This basal current displayed a linear I-V relationship with a reversal potential close to 0 mV (Fig. 1C) and was virtually absent when ATP was omitted from the pipette (97 ± 41 pS, n = 4 healthy individuals) (Fig. 1D). The anion permeability of this pathway, determined by the observed shift in reversal potential (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google a sequence of The current was addition of the (1 μm) to the bath (Fig. and was by the chloride channel niflumic acid (100 μm) and glibenclamide (100 μm) K. PubMed Scopus Google Scholar). the chloride channel inhibitor μm), as well as the new permeation furosemide (100 μm) (1Kirk K. Physiol. Rev. 2001; 81: 495-537Crossref PubMed Scopus (328) Google Scholar, S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar, H. PubMed Scopus Google had no on this basal current. of to the or addition of the against the CF-2 of CFTR G. P. J. 1992; Full Text PDF PubMed Google Scholar) to the of the in strong the low current in CF red blood cells. The effect was by the CF-2 as (Fig. the of the effect of the in the of the chloride conductance on ATP and PKA, by and absence from CF red blood cells, support the that CFTR chloride channels are present in red blood cells and are under This concept is by the on the of RT-PCR, of low levels of CFTR in cells, the isolated from bone marrow from healthy individuals (Fig. and by the recent of a linear anion conductance in uninfected non-CF red blood cells G. Staines H.M. C. J. Physiol. Scopus Google Scholar). data for the first indicate a functional expression of CFTR in control but not in CFTRΔPhe-508/ΔPhe-508 red blood cells. of the erythrocytes by by the osmolarity from the bath from 300 to by increasing the mannitol in the of an additional chloride conductance (Fig. of similar and with an identical I-V relationship (Fig. pharmacological profile (Fig. and anion sequence to that for the P. inwardly rectifying chloride current (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar). to the CFTR-like this conductance was on ATP and with all CFTR tested the G449 and (Fig. from the CFTR-like current in to a inhibitor of the new permeation pathway and of the Plasmodium-activated chloride conductance (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar). The furosemide to as an inhibitor of the to 250 μm), a was to the chloride current not this inwardly rectifying chloride current was in red blood cells from CF patients (Fig. a close functional relationship with the CFTR chloride basal conductances by whole-cell in medium were not different between control and CF red blood cells not against a effect of the CF on of the red blood cell To the pharmacological of the and the malaria-induced chloride we performed patch clamp experiments on P. falciparum red blood cells from healthy individuals at with the study by (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google in the of an inwardly rectifying chloride current that was under isosmotic conditions (Fig. The and I-V relationship (Fig. of this malaria-induced chloride conductance were identical to the endogenous chloride Moreover, shrinkage of infected red blood cells did not the chloride conductance of the membrane (Fig. that the malaria-induced current and the cell endogenous current are of the inwardly rectifying chloride the pharmacological profile (Fig. identical to that of the shrinkage-activated chloride conductance (Fig. that the conductances in at least shrinkage of the chloride channel is on ATP and PKA and by the P. channel in the absence of ATP (Fig. (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar) and is to and G449 (Fig. that the of the of CFTR by PKA is necessary for shrinkage of the inwardly rectifying chloride channel but not for P. falciparum of the of red blood cells with P. falciparum in a of CFTR, by of endogenous Plasmodium PKA 2000; PubMed Scopus Google Scholar) or protein kinase W. J. PubMed Scopus Google which is in the of the parasite W. PubMed Scopus Google Scholar). The protein kinase was demonstrated to be in the in intracellular survival of the parasite J. B. J. Cell. PubMed Scopus Google Scholar). A role of protein in chloride channel is also by the that the Plasmodium-activated chloride current when acid phosphatase is added to the pipette (n = 4) (Fig. current was observed when acid phosphatase was added to the pipette (n = 4) (Fig. also stage cells h as by following with the (Fig. was no in of the inwardly rectifying chloride channel compared with cells (Fig. that of the channel is at an stage of of the new permeation pathway (n = 4) H.M. K. Am. J. Physiol. 2001; PubMed Google of infected non-CF erythrocytes to acid phosphatase (30 units/ml) in the pipette in the absence of The rectifying current no current was with of stage infected cells with the of the red blood cells The indicate infected cells, which be used for patch clamp of the Plasmodium-activated inwardly rectifying chloride current in a stage The rectifying current is the new permeation pathway CF red blood cells, CFTR and offered us the unique possibility to study the obligatory role of this chloride channel in the survival of P. falciparum. the channel is identical to the endogenous shrinkage-activated channel, as suggested by the experiments and the new permeation pathway is the of this channel as suggested by (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google then the new permeation pathway, and thus survival of the malaria be to be in CF red blood cells. To this blood samples from CFTRΔPhe-508/ΔPhe-508 patients and were used for in vitro culture of P. falciparum FCR3 and NF54 strains were used to the of [3H]hypoxanthine incorporation no in invasion and development of the P. falciparum FCR3 parasites be observed between and CFTRΔPhe-508/ΔPhe-508 red blood cells (Fig. of the cultures by of Giemsa-stained thin demonstrated that parasites in all cultures and had growth of the CF were obtained with the NF54 parasite patch clamp of the malaria-infected CF red blood cells that the parasites had to the inwardly rectifying current (Fig. that the chloride channel is not obligatory for parasite survival in To the functional absence of the inwardly rectifying channel the activity of the new permeation pathway, we performed isotonic lysis experiments in red blood cells to the of choline chloride, glucose, and alanine. In experiments the infected red blood cells are incubated in an isosmotic solution of of the movement of the through the red blood cell membrane and lysis of the The lysis be used to the new permeation pathway activity (1Kirk K. Physiol. Rev. 2001; 81: 495-537Crossref PubMed Scopus (328) Google Scholar) and thus to in nutrient the red blood cell membrane of CF and non-CF cells. The observed between infected non-CF and CF red blood cells was at the of glucose uptake for all n = 4) (Fig. experiments suggested that the of alanine and choline is in infected CF red blood cells, the percentage of lysis was similar in infected CF and non-CF red blood cells. data that the inwardly rectifying chloride channel accounts for a of the nutrient in infected red blood cells. data indicate that the of nutrients in infected CF red blood cells had no on parasite growth under standard in vitro culture as by the experiments (Fig. the new permeation furosemide glucose and alanine and choline both in infected control and CF red blood cells not indicate that the of furosemide be to the of the inwardly rectifying chloride channel as this channel is in non-CF cells. This is by the that parasites in CF red blood cells to the new permeation pathway as demonstrated by the in parasitemia in both CF and non-CF red blood cells following a to furosemide (Fig. The that a CFTR-like chloride conductance is in but not in CF red blood cells. with data on CFTR expression in red blood cell cells, as well as on of channel by a study for the first that CFTR is for an endogenous chloride conductance in red blood cells. Moreover, also indicate that CFTR is for the of a inwardly rectifying chloride current by cell shrinkage or P. falciparum the inwardly rectifying conductance from the channel with respect to several and pharmacological properties permeability rectifying in the absence of and furosemide data support a channel in which CFTR and the but are to the well relationship between CFTR and the rectifying chloride channel K. H. N. J. PubMed Scopus Google Scholar). in of recent data that and of CFTR in from those in cultured cells Nature. PubMed Scopus Google the present evidence for a than is and of the inwardly rectifying at the data not support the by (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar) that the Plasmodium-activated inwardly rectifying channel B. S.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar, S.A. PubMed Scopus Google accounts for the parasite-induced in permeability to a of was on the similar pharmacological profile of the inwardly rectifying chloride conductance and the new permeation pathway (6Desai S.A. Bezrukov S.M. Zimmerberg J. Nature. 2000; 406: 1001-1005Crossref PubMed Scopus (217) Google Scholar) and on the that the chloride of infected red blood cells, as from patch clamp analysis, the chloride conductance from chloride measurements B. S.A. J. Full Text Full Text PDF PubMed Scopus Google Scholar). data a role for the inwardly rectifying chloride This is from the development of malaria parasites in CF red blood cells, in which the inwardly rectifying chloride conductance is and from the of the CF on of the parasite-induced new permeation pathway, that the is not the target for the of stage cells demonstrated that the inwardly rectifying chloride conductance is the new permeation pathway (Fig. and the of the parasite-induced inwardly rectifying chloride conductance following protein by acid a protein for the first that the of the inwardly rectifying chloride conductance be This that the inwardly rectifying chloride conductance is through of red blood cell and against potential of channel or protein as was suggested by Huber S.M. C. J. PubMed Scopus Google Scholar). were also to an anion channel with rectifying properties that is by Plasmodium was demonstrated that the rectifying current is when the holding potential is negative or when human is added to the bath H.M. G. C. Huber S.M. J. Physiol. Scopus Google Scholar, J. C. Huber K. K. PubMed Scopus Google Scholar). In we a holding potential of 0 and the human in the a from the human necessary for the Plasmodium is than Although we that the inwardly rectifying chloride conductance is identical to the endogenous shrinkage-activated inwardly rectifying chloride and than data not the new permeation pathway is endogenous or and not a as to the identity of the malaria-induced new permeation pathway. be that the against a role of CFTR and the inwardly rectifying chloride conductance in new permeation pathway not the possibility that chloride channel in red blood cells of under in that channels are defective in CF the to this in CF the functional expression of CFTR in red blood cells opens new perspectives to exploit the erythrocyte as a readily available cell type in electrophysiological, diagnostic, and therapeutic studies of CF. M. Lindern is for the human