enhanced lysosomal acidification leads to increased chloroquine accumulation in cho cells expressing...

11
ELSEVIER Molecular and BiochemicalParasitology68 (1994) 209-219 MOLECULAR AND BIOCHEMICAL PARASITOLOGY Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdrl gene Helmuth H.G. van Es a,1 Herma Renkema b Hans Aerts b, Erwin Schurr a,, 9 a Department of Medicine, McGill University, MontrEal, Canada b The E.C. Slater Institute for BiochemicalResearch, University of Amsterdam, Amsterdam, The Netherlands Received 4 May 1994; accepted 25 August 1994 Abstract Expression of the pfmdrl-encoded Pghl protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pghl-mediated accumulation of this antimalarial. Pghl carrying amino acid substitutions associated with chloroquine resistance in P. falciparum does not confer this phenotype. Here, we present studies on the underlying mechanism of Pghl mediated chloroquine influx into CHO cells. First, we measured intralysosomal pH using FITC-labelled dextran and found the intralysosomal pH in Pghl expressing cells to be decreased. A decreased lysosomal pH was not observed in cells expressing Pghl carrying the S1034C and N1042D double substitution found in some chloroquine-resistant P. falciparum parasites. Secondly, Pghl-mediated uptake of chloroquine was abolished in the presence of bafilomycin A1, a specific inhibitor of vacuolar [H + ]ATPases and was nearly abrogated in the presence of NHaC1. Finally, cells expressing wild-type Pghl showed increased uptake of both (+)- and (-)[3H]chloroquine enan- tiomers, indicating that Pghl-mediated uptake of chloroquine is not enantioselective and in agreement with a pH-driven process. We conclude from these studies that Pghl does not transport chloroquine, but instead influences chloroquine accumulation by modulating the pH of acidic organelles. This function is abolished in Pghl carrying amino acid substitutions S1034C and N1042D. We speculate that the pfmdrl gene encodes a vacuolar chloride channel. Keywords: Plasmodium falciparum; Chloroquineresistance; P-glycoprotein; Expression system Abbreviations: Pgps, mammalian P-glycoproteins; CQ, choloroquine; CQR chloroquine-resistant; CQS chloroquine-sensi- tive * Corresponding author. Present address: Montr6al General Hospital, Centre for Host Resistance, 1650 Cedar Avenue, H3G 1A4, Montr6al, Qu6bec, Canada. Tel: (514)-937 6011, Ext. 4554; Fax: (514)-934 8261; [email protected]. 1Present address: Introgene BV, Postbus 3271, 2280 GG Rijswijk, The Netherlands. 1. Introduction The pfmdrl gene product is part of a large group of membrane proteins which are involved in trans- membrane transport. Members of this family have normally at least 6 transmembrane spanning do- mains, a cytoplasmic ATP binding cassette (ABC) 0166-6851/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0166-6851(94)00164-2

Upload: helmuth-hg-van-es

Post on 22-Aug-2016

216 views

Category:

Documents


4 download

TRANSCRIPT

Page 1: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

E L S E V I E R Molecular and Biochemical Parasitology 68 (1994) 209-219

MOLECULAR AND BIOCHEMICAL PARASITOLOGY

Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdrl gene

H e l m u t h H.G. van Es a,1 H e r m a R e n k e m a b Hans Aerts b, Erwin Schurr a,, 9

a Department of Medicine, McGill University, MontrEal, Canada b The E.C. Slater Institute for BiochemicalResearch, University of Amsterdam, Amsterdam, The Netherlands

Received 4 May 1994; accepted 25 August 1994

Abstract

Expression of the pfmdrl-encoded Pghl protein of Plasmodium falciparum in CHO cells confers a phenotype of increased sensitivity to chloroquine due to an increased Pghl-mediated accumulation of this antimalarial. Pghl carrying amino acid substitutions associated with chloroquine resistance in P. falciparum does not confer this phenotype. Here, we present studies on the underlying mechanism of Pghl mediated chloroquine influx into CHO cells. First, we measured intralysosomal pH using FITC-labelled dextran and found the intralysosomal pH in Pghl expressing cells to be decreased. A decreased lysosomal pH was not observed in cells expressing Pghl carrying the S1034C and N1042D double substitution found in some chloroquine-resistant P. falciparum parasites. Secondly, Pghl-mediated uptake of chloroquine was abolished in the presence of bafilomycin A1, a specific inhibitor of vacuolar [H + ]ATPases and was nearly abrogated in the presence of NHaC1. Finally, cells expressing wild-type Pghl showed increased uptake of both (+) - and (-)[3H]chloroquine enan- tiomers, indicating that Pghl-mediated uptake of chloroquine is not enantioselective and in agreement with a pH-driven process. We conclude from these studies that Pghl does not transport chloroquine, but instead influences chloroquine accumulation by modulating the pH of acidic organelles. This function is abolished in Pghl carrying amino acid substitutions S1034C and N1042D. We speculate that the pfmdrl gene encodes a vacuolar chloride channel.

Keywords: Plasmodium falciparum; Chloroquine resistance; P-glycoprotein; Expression system

Abbreviations: Pgps, mammalian P-glycoproteins; CQ, choloroquine; CQR chloroquine-resistant; CQS chloroquine-sensi- tive

* Corresponding author. Present address: Montr6al General Hospital, Centre for Host Resistance, 1650 Cedar Avenue, H3G 1A4, Montr6al, Qu6bec, Canada. Tel: (514)-937 6011, Ext. 4554; Fax: (514)-934 8261; [email protected].

1Present address: Introgene BV, Postbus 3271, 2280 GG Rijswijk, The Netherlands.

1. Introduction

The pfmdrl gene product is part of a large group of membrane proteins which are involved in trans- membrane transport. Members of this family have normally at least 6 transmembrane spanning do- mains, a cytoplasmic ATP binding cassette (ABC)

0166-6851/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0166-6851(94)00164-2

Page 2: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

210 H.H.G. uan Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219

characterized by Walker A and B motifs, and the ABC signature sequence [1]. Most ABC proteins are known to be implicated in the ATP-dependent trans- port of a variety of substrates, ranging from polypep- tides (TAPI/2) [2,3], chloride ions (CFTR) [4] and phospholipids (mdr2) [5] to metals such as Cd (HMT1) [6]. The pfmdrl and HMT1 gene products, distinguish themselves from other known ABC trans- porters by their intracellular localization since they are both found in the membrane of vacuolar com- partments. In the case of pfmdrl this is the food vacuole of Plasmodiumfalciparum an organelle with lysosomal features, namely acidic pH and a high content of hydrolases with acidic pH optimum [7-9]. The pfmdrl-encoded polypeptide Pghl displays a predicted topology highly similar to mammalian P- glycoproteins (Pgps), i.e., the structural unit contain- ing the three transmembrane loops and the ABC motif has been duplicated and the duplicated halves have been fused via a hydrophilic linker region [5].

Plasmodium parasites accumulate the antimalarial drug chloroquine (CQ) approximately 800-fold over their surrounding extracellular environment in the lumen of the digestive vacuole. It is generally ac- cepted that this accumulation forms the basis of the parasiticidal action of CQ, since CQ-resistant (CQR) parasites accumulate less CQ than CQ-sensitive (CQS) parasites [10-12]. Earlier studies have sug- gested that the basis of the CQR phenotype in certain parasite isolates is an enhanced effiux of CQ which can be reversed by verapamil. The increased CQ effiux results in a lowered steady-state level of intra- cellular CQ and hence CQR [7,11,13,14]. This mech- anism of drug resistance is analogous to the one displayed by multidrug-resistant cancer cells which overexpress the Pgp ABC drug transporter [1,15]. Pgp overexpressing cells are drug-resistant due to a Pgp-mediated, energy-dependent and verapamil-re- versible drug extrusion mechanism. It was therefore reasoned that a plasmodial Pgp homologue is in- volved in CQR, a conclusion which led to the isola- tion of the pfindrl gene [7]. Mutations in the pfmdrl gene in many geographical isolates are strongly asso- ciated with the CQR phenotype [16]. However, more recent studies have put in question the association between CQR, specific pfmdrl mutations and the increased CQ effiux phenotype [17-19].

Previous studies on P. falciparum parasites sug-

gested that the pH of the digestive vacuole is ele- vated in CQR parasites. Because CQ is a weak base, an increased vacuolar pH would result in lower CQ levels in this vital organelle. Lower vacuolar concen- trations of CQ will decrease inhibition of the target of CQ, the heine polymerase [20-24]. A rise in baseline pH of the food vacuole in CQR parasites is also supported by more recent data showing that CQR parasites are more sensitive to bafilomycin A1 a specific inhibitor of vacuolar [H+]ATPases. These results indicate that vacuolar acidification is de- creased either due to a weakened [H+]ATPase [12] or perhaps due to a reduced activity of a vacuolar chloride channel. No structural differences were ob- served in the [H+]ATPase subunit A of CQS and CQR parasites suggesting that a mutated [H +]ATPase is not the cause of changes in vacuolar pH [25].

If the increase in the steady-state vacuolar pH is critical in establishing resistance to CQ, it is possible that the pfmdrl-encoded protein Pghl plays a role in the regulation of the pH of the digestive vacuole. We have previously employed heterologous expression in CHO cells to study the function of the pfmdrl-en- coded protein, Pghl. We have demonstrated that the wild-type Pghl protein as found in chloroquine-sen- sitive (CQS) parasites (e.g., D10 isolate) mediates verapamil-insensitive susceptibility to chloroquine due to an increased, Pghl-mediated accumulation of CQ. Mutant pfmdrl carrying the CQR associated allele as found in P. falciparum isolate 7G8, does not confer this phenotype [16,26]. These results strongly suggest a role for the pfmdrl gene in the CQR phenotype of P. falciparum and open the possibility that Pghl is involved in the regulation of the vacuolar pH.

In this report we have addressed the hypothesis of Pghl-mediated regulation of the intralysosomal pH by measurement and modulation of the lysosomal pH in cells expressing Pghl. For this purpose we employed the [H+]ATPase inhibitor bafilomycin A1 and the vacuologenic amine NH4CI and measured the lysosomal pH by using FITC-dextran labelling. To find evidence for carrier-mediated transport we employed purified enantiomers in CQ accumulation studies. The results of these experiments suggest that pfmdrl does not encode a CQ carrier but instead encodes a protein which modulates the low pH of acidic organelles.

Page 3: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

H.H.G. van Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219 211

2. Materials and methods

2.1. Transfections and cell cultures

Pghl-expressing CHO cells were obtained by transfection of DHFR deficient DUK cells with the pfmdrl gene cloned into pEMC2 as described previ- ously. The expression levels of Pghl in these trans- fectants were similar, as determined by Western blotting [26]. Stable transfectants were grown in a-MEM medium minus purines and pyrimidines/ 10% dialyzed fetal calf serum/20 nM methotrexate (MEM/MTX).

2.2. Determination of intralysosomal pH using FITC-dextran labelling

Prior to pH determination pfindrl transfectants grown to approximately 80% confluency were trans- ferred to medium containing 0.4 mg m1-1 FITC-de- xtran (Sigma, molecular size range 70 kDa). The next day, FITC-dextran was chased into lysosomal compartments by incubating the cells in normal M EM/MTX medium for 30 min. Cells were then harvested by trypsinization, washed once with buffer A (140 mM NaCI/1 mM CaC12/1 mM MgCI2/10 mM glucose/25 mM sodium acetate, pH 7.5) and finally resuspended in 2 ml of prewarmed (37°C) buffer A in a fluorimeter cuvette. Immediately after resuspension the fluorescence ratio at 495/450 nm was measured in a LS50 Perkin-Elmer luminometer. After registration of the initial ratio monensin was added to a final concentration of 5 /xM resulting in an increase of the ratio equivalent to the pH of buffer A. The pH was then titrated to a fluorescence ratio equal to the initial value before addition of monensin by addition of 10% acetic acid. After every addition of acetic acid the pH was measured in the cuvette. The method described here is a modification of the method originally described elsewhere [27].

2.3. HPLC purification of enantiomers of chloro- quine

Tritiated racemic CQ dissolved in ethanol was purchased from Amersham, PLC (specific radioactiv- ity = 4.9 Ci mmol 2). Ethanol was removed by evaporation in a Speedvac followed by dissolving the racemic mixture of [3H]CQ in 30 mM sodium

phosphate buffer, pH 7.0/ethanol/acetonitr i le (79:20:1, v / v / v ) containing 5 mM N,N-dimethyl- octylamine (N,N-DMOA), which served as the mo- bile phase for enantioselective chromatography. HPLC separation of the two [3H]CQ enantiomers was carried out at ambient temperature on a column packed with a chiral stationary phase (CSP) consist- ing of immobilized as-acid glycoprotein and a Chi- ral-AGP guard column (Regis Chemical Co.) [28]. Several rounds of purification were carried out and the enantiomer peaks were pooled. Pooled purified (+ ) - or ( - ) - [3H]CQ enantiomers were alkalinized with NaOH and the CQ free base was extracted into diethylether by vortexing for 1 min followed by centrifugation for 10 min at 1750 × g. After centrifu- gation the aqueous phase was frozen by placing the tubes in a slurry of dry ice and acetone. The organic phase was collected and the extraction repeated with the remaining aqueous phase. Then the diethylether was evaporated and the purified [3H]CQ enantiomers were dissolved in ethanol. Aliquots of ( + ) - or ( - ) - [3H]CQ enantiomer were quantitated on the Chiral- AGP column with unlabelled racemic CQ as a stan- dard.

2.4. Accumulation of [3H]CQ in pfmdrl transfec- tants

The CHO cells were seeded in 24-well plates at a cell density of 0.6-1 × 105 cells per well. The fol- lowing day the medium was removed and the cells washed with 2 ml PBS (Dulbecco's plus 0.5 mM MgCI2/0.9 mM CaCI 2) at ambient temperature. The cells were then incubated with 0.4 ml PBS with 10 mM glucose for 15 min. At t = 0 min 100 /xl of [3H]CQ ([3H]CQ] = 20-40 nM, specific radioactiv- ity = 4.9 Ci mmol -~) in PBS with 10 mM glucose was added. Uptake was stopped by adding 2 ml of cold PBS after indicated periods of time. Then, PBS was removed, cells were trypsinized followed by solubilization with 0.125% Triton-X-100 and spun for 10 min at maximum speed in a microcentrifuge. Aliquots of supernatants were counted in a liquid scintillation counter. Results of CQ uptake are ex- pressed as pmol CQ (mg total cellular protein) -~. The difference in [3H]CQ accumulation between Pghl wild-type transfectants and controls was highly significant (P < 0.001) [26].

Page 4: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

212 H.H.G. van Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219

3 . R e s u l t s

3.1. lntralysosomal pH in Pghl-expressing cells

The phenotype of Pghl-expressing CHO cells is characterized by a specific increase in sensitivity to the antimalarial CQ. This increase is due to an enhanced, Pghl-mediated, energy-dependent uptake of CQ [26]. In mammalian cells, CQ, a diprotic weak base, accumulates in acidic organelles like lysosomes due to trapping of protonated CQ [29]. Thus, one possible explanation for the observed increased up- take in Pghl-expressing cells is that the pH of acidic organelles is decreased due to increased Pghl-media- ted acidification. We measured the intralysosomal pH by labelling the lysosomes with FITC-dextran and subsequently determined the pH dependent fluo- rescence ratio of FITC. This method was previously used by other investigators to determine intralysoso- mal pH [27]. In control cells the pH values ranged from 5.0 to 5.4 with a mean value of 5.2 + 0.1 (SE, n = 9)). In cells expressing mutant Pghl (S1034C/N1042D) the pH varied from 5.0 to 5.4 with a mean value of 5.2 + 0.1 (n = 5). In contrast, in cells expressing wild-type Pghl the pH was clearly more acidic, ranging from pH 4.5 to 5.0 (Fig. 1). This pH decrease was observed in two cell lines expressing wild-type pfmdrl. These transfectants were derived from two independent transfections. The mean lysosomal pH value for these two transfec- tants is 4.8 + 0.1 (n = 7). The absence of a decrease in lysosomal pH in cells expressing the mutant Pghl is in close agreement with the absence of increased CQ sensitivity and accumulation in these cells (Fig. 4) and [26].

3.2. Inhibition of [H +]ATPase by bafilomycin A1 in Pghl-expressing cells

The vacuolar electrogenic [H+]ATPase found in the membrane of organelles such as lysosomes and endosomes is the major determinant of the low pH in both normal and Pghl-expressing CHO cells. Spe- cific inhibition of [H+]ATPases allows the distinc- tion between direct Pghl-mediated CQ transport and CQ transport that is solely dependent on the pH gradient that exists between the cytosol and acidic

"I-

E o o

.-I

5.4

5.2

5.0

4.8

4.6

4.4

Fig. 1. Lysosomal pH in cells expressing Pghl. Pfmdrl transfec- tants were loaded with FITC-dextran and subjected to in vivo ratio fluorimetric measurements as described in Materials and methods and [27]. The differences in pH values between control cells and wild-type Pghl transfectants were statistically highly significant (P < 0.001). Data are given as unaveraged values and are the result of multiple individual experiments determined on different days. (©), antisense control transfectants which do not express Pghl; (0) and ( I ) , (P5) and (R4) respectively, transfectants which both express CQS Pghl. P5 and R4 are derived from two independent transfections; (0) transfectants expressing a CQR Pghl with a double amino acid substitution, S1034C and N1042D.

organelles. This is possible with bafilomycin A1, a specific inhibitor of vacuolar [H+]ATPases in a wide variety of organisms including mammalian cells and P. falciparum [12,30,31]. Preincubation of pfmdrl transfectants in the presence of 1 /~M bafilomycin A1 before addition of [3H]CQ decreases the total cellular CO accumulation significantly. After approx- imately 10 min of incubation with [3H]CQ a plateau in CQ uptake is reached at 20-30% of the initial values observed without bafilomycin A1. Impor- tantly preincubation with bafilomycin A1 completely removes the Pghl-mediated component of CQ-up- take (Fig. 2). Residual accumulation levels in the presence of bafilomycin A1 probably represent total cellular accumulation which does not involve acidic organelles although incomplete inhibition of the vac- uolar [H+]ATPase can not be excluded. The mor- phology of the cells was monitored by microscopic viewing of the cells during the course of the incuba- tions. No abnormalities were observed. These results

Page 5: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

H.H.G. van Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219 213

indicate that the pH is a major component of the Pghl -media ted CQ accumulation and they are in line with the observed increased lysosomal acidification in cells that express wild type Pghl (Fig. 1).

3.3. Effect o f ammonium chloride on chloroquine accumulation in Pghl-express ing cells

Instead o f b l o c k i n g the vacuolar [H + ]ATPase, the main protein responsible for generating the lysoso- mal pH gradient, it is important to know what hap- pens with the Pghl -media ted CQ uptake upon col- lapsing the pH gradient that exists over the lysoso- mal membrane, while leaving the [H+]ATPase ac- tive. This can be done by using the vacuologenic amine NH4C1 which has been shown to be an effec- tive agent for alkalinization of the lysosomal lumen [29]. To address the effect of alkalinization on CQ accumulation in Pghl -express ing cells, the experi-

]00 l " I I I

1:3

o

~ ~ 150

N 100 8 "~.

50 ,i._____...__._, °

0 I I I 20 40 GO

T i m e in m i n u t e s

Fig. 2. Accumulation of [3H]CQ in the presence of bafilomycin A1 in Pghl-expressing cells. Cells were preincubated in PBS, 10 mM glucose without (O,D,~) or with (O, II ,O) 1 mM bafilomycin A1 before the addition of [3H]chloroquine. ( 0 ,0 ) , antisense control transfectants which do not express Pghl; (D, • ) sense transfectants expressing CQS Pghl (P5); (~, • ) sense trans- fectants expressing mutant CQR Pghl with amino acid substitu- tions S1034C and N1042D. The graph presented is derived from one representative experiment in which each time point was measured in duplicate. Similar results were obtained in 4 addi- tional experiments.

18

l,° ffl

' ~ 14

% "C

& 12

o IO E 8 "~..

8 ._= O

1 3 5 7 10

[NH4CI ] in mM

Fig. 3. Effect of NH4CI on the accumulation of CQ in pfmdrl-ex- pressing cells. Before initiating CQ uptake the cells were preincu- bated for 10 rain in the presence of increasing concentrations of NH4CI in PBS and 10 mM glucose. Blank bars (W3), nonexpress- ing control; shaded bars (R4) and black bars (P5) are both wild-type Pghl-expressing cells. The graph presented is derived from one representative experiment in which each point was measured in duplicate. Similar results were obtained in 3 addi- tional experiments.

ments were done in the presence of varying NH4C1 concentrations. Preincubation of cells with increasing concentrations of NHaC1 leads to an exponential decline in the accumulation of [3H]CQ in both con- trol and Pghl -express ing cells. Only at the highest NHaCI concentrations used is the Pghl -media ted component of CQ accumulation decreased to a value near but not equal to that observed in non-expressing cells (Fig. 3). The inverse exponential relationship between CQ accumulation and the NH4CI concentra- tion suggests that at infinite concentrations of NH4C1 the residual difference between control and express- ing cells would completely disappear. These experi- ments confirm that the pH is a major driving force for CQ accumulation in both control cells and cells expressing wild-type Pghl . Since the Pghl -media ted component of CQ-uptake is abrogated by addition of NH4C1 it is l ikely that Pgh l causes increased acidifi- cation of lysosomes. This conclusion is in agreement with the lysosomal pH determinations and the baf i lomycin A1 experiments (Fig. 2).

Page 6: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

214 H.H.G. uan Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219

3.4. Accumulation of purified enantiomers of [3H]chloroquine in Pghl-expressing cells

To further investigate whether the Pghl-mediated CQ accumulation in CHO cells is a pH gradient driven process or a carrier-mediated transport, we purified the two tritiated enantiomers of CQ by chiral HPLC from a commercially available racemic mixture. A stereoselective influx would support a carrier-mediated process. Using the HPLC system described in Materials and methods the retention time for the (-)-enantiomer was found to be 16.5 min and for the (+)-enantiomer 19 min (Fig. 4A). This is in close agreement with previous studies using the same HPLC setup and conditions for the separation of unlabelled CQ enantiomers [28]. This difference in retention time enabled us to separate the two CQ enantiomers on an analytical scale and to obtain sufficient quantities of each enantiomer for uptake experiments. The purified enantiomers were used to study CQ accumulation in three different types of pfmdrl transfectants: cells that do not ex- press Pghl, cells expressing wild-type (CQS) Pghland cells expressing Pghl carrying the double mutant allele S1034C/N1042D. The results of these

experiments are expressed as -fold uptake of CQ over nonexpressing control cells (Fig. 4B). These data show that in cells expressing wild-type Pghl the enhanced uptake of CQ is not different for the two CQ enantiomers. Importantly, cells expressing mu- tant Pghl do not show an increased uptake of either CQ enantiomer as has been described for the racemic mixture of [ 3 H ] C Q [26].

4. Discussion

Many explanations have been presented for the CQR phenotype of P. falciparum parasites. In- creased verapamil-sensitive efflux of CQ has been described for the CQR strains IC1 and Dd2 as compared to the CQS strains Haiti 135 and HB3 [11,13,32]. These data suggested that a homologue of the mammalian P-glycoproteins was involved in the expression of the CQR phenotype, in analogy to multidrug-resistant cancer cells [11,13]. The cloning of the pfmdrl gene and the subsequent finding that specific alleles of this gene closely associate with the CQR phenotype in many strains indeed indicate a

A

g E

B

(-)-enantiorner, I= 16.5 rnin

(+)-enantiomer, I=19 rain

o

o ~ 1 4 o .~-

g=

o m

N

1 2 3

T i m e in m i n u t e s ~ ( + ) - C h l o r e q u i n e

+t

1 3

(-)-Chloroquine

Fig. 4. (A) Separation of the two [3H]chloroquine enantiomers on a Chiral-AGP HPLC column. Relative retention times for each enantiomer are indicated. For details of HPLC conditions see Materials and methods. (B) Accumulation of [3H]CQ enantiomers in cells expressing wild-type or mutant pfmdrl. Cells ( 6 - 1 0 X 104) were seeded in 24-well plates and incubated with approximately 40 nM (+ ) - or ( - ) - [3H]CQ for 90 min before stopping uptake with cold PBS. (1) and (2): R4 and P5, wild-type Pghl-expressing cells derived from two independent transfections and (3) cells expressing Pghl carrying the S1034C and N1042D double substitution. Data are expressed as -fold accumulation values over nonexpressing W3 cells and are the mean of 3 -4 independent experiments.

Page 7: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

H.H.G. van Es et al. / Molecular and Biochemical Parasitology 68 (1994) 209-219 215

role for the pfmdrl gene in the drug resistance of P. falciparum [7,16]. However, in more recent CQR isolates, no amino acid changes in the pfmdrl gene were found [18,19]. In addition, a general explana- tion of drug resistance by increased efflux of CQ is at odds with studies on other CQR parasites, T9-94 and K1 which were compared to CQS isolates 3D7 and T9-96. These CQR isolates did not exhibit an increased efflux rate of CQ. Based on these results it was postulated that the capacity to concentrate CQ in the digestive vacuole is changed in CQR parasites [17]. Similar suggestions had previously been made by other investigators who analysed the kinetics of CQ uptake and efflux in CQS and CQR P. falci- parum parasites [21,33]. It was concluded from these studies that a CQ concentration mechanism in CQR parasites is changed either by an alteration of the vacuolar pH or by a lowered activity of a putative vacuolar CQ importer. Experimental evidence has now emerged that suggests that the steady-state vac- uolar pH of CQR parasites is increased, explaining lowered accumulation of CQ in CQR isolates [12]. Unfortunately, a comprehensive detailed comparison between the vacuolar pH of CQS and CQR parasites has not been performed. Nevertheless, in vitro stud- ies using crude membrane vesicles of CQS (Haiti 135) and CQR (IC1) parasites suggested that indeed in the CQR isolate used, an acidification factor is decreased in capacity [34].

We recently developed a heterologous expression system for the pfmdrl gene which enabled us to show that the pfmdrl-encoded protein, Pghl medi- ates increased CQ sensitivity of CHO cells due to increased Pghl-mediated CQ accumulation. This phenotype is not conferred by the pfindrl gene carrying CQR mutations [26]. The CQ sensitivity of pfmdrl-expressing CHO cells is verapamil insensi- tive which contrasts with the observed partial rever- sal of CQR by verapamil in P. falciparum [11,13]. This difference between Pghl-expressing CHO cells and Plasmodium suggests that CQR in Plasmodium parasites involves complex biochemical interactions which are not exclusively determined by the pfmdrl gene. Indeed, several findings strongly suggest that the CQR phenotype of P. falciparum is a multigenic trait. Wellems et al., have shown that on the basis of a genetic cross between CQR and CQS parasites pfmdrl does not link with the CQR phenotype [32]

and mapped another CQR locus to chromosome 7 of P. falciparum [35]. In addition, a recent report has shown that some CQR isolates have increased levels of pfmdr2 transcripts suggesting a potential role for this gene in drug resistance [36]. The latter report opposes the findings of Wellems and colleagues who concluded that pfmdr2 is not important for CQR on the basis of their genetic cross [32]. Taken together these data clearly indicate that the CQR phenotype is a complex trait. As P. falciparum isolates of differ- ent geographic origins are being compared it is likely that genetic heterogeneity and epistasis need to be considered to achieve a full understanding of the trait. In addition, it is conceivable that environmental factors such as local climate and compliance with treatment protocols may further influence expression of CQR in P. falciparum.

In the studies reported here we have investigated whether Pghl modulates the acidification of lyso- somes and if so whether there is a difference in lysosomal acidification between transfectants ex- pressing wild-type (CQS) or CQR-associated mutant Pghl. The expression of the CQS-associated Pghl in mammalian cells results in a phenotype characterized by increased uptake of CQ and increased CQ sensi- tivity [26]. The results presented here suggest that Pghl-mediated uptake of CQ can be explained by Pghl dependent modulation of the intralysosomal pH. This conclusion is based on several independent lines of experimental evidence: First, evidence for lysosomal localization of Pghl expressed in CHO cells is provided by a specific increase in inhibition of the CQ sensitive, lysosomal a-galactosidase in Pghl-expressing cells and immunolocalization exper- iments [26]. Preliminary Percoll fractionation experi- ments confirm that Pghl is present in lysosomes, endosomes and possibly the ER (van Es and Schurr, unpublished observations). Secondly, Pghl-mediated uptake is not different for the two enantiomers of CQ (Fig. 4). This suggests that CQ accumulation is a non-stereoselective process. Although the number of studies in which the stereoselectivity of transmem- brane transport was investigated is limited, there are well-described transporters that show a high degree of selectivity for only one of the two enantiomers. Examples are the glucose and alanine transporters of renal brush border membranes [37] and the murine mdrl protein-mediated resistance to gramicidin,

Page 8: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

216 H.H.G. van Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219

which is stereoselective for the D-enantiomer [38]. However, lack of enantioselectivity of Pghl-media- ted CQ uptake is merely suggestive for a non- carrier-mediated uptake and therefore we cannot firmly rule out some degree of carrier-mediated transport by Pghl. Further support for a pH-driven event is given by our experiments in which CQ uptake is reduced by 70-80% in both Pghl-ex- pressing and non-expressing cells by NH4C1 treat- ment. However, Pghl-expressing cells consistently accumulate higher CQ levels over non-expressing cells. The reason for this residual differential CQ accumulation in the presence of NH4C1 is not clear. It is possible that a residual Pghl-mediated pH dif- ference between control and expressing cells still exists under the conditions used for alkalinization of acidic organelles. Other evidence for a pH driven process comes from experiments in which cells were preincubated in the presence of bafilomycin A1. Bafilomycin A1 which is a potent specific inhibitor of the vacuolar [H+]ATPases [30,31] completely re- moves the difference in CQ uptake between express- ing and control cells (Fig. 2). If the Pghl-mediated CQ uptake would have been due to direct transloca- tion of CQ by Pghl the increase in CQ uptake should have remained unchanged in the presence of bafilomycin A1. These data do not exclude the hypo- thetical scenario in which Pghl employs the energy of the pH gradient to drive transport of CQ. A number of transporters are known that use the pH gradient over the mammalian lysosomal membrane to drive transport, including the sialic acid trans- porter [39]. Nevertheless, pH-gradient coupling to CQ transport appears unlikely since Pghl, like other ABC transporters, is expected to use ATP to drive CQ transport. Indeed, under conditions of ATP de- pletion a decrease of Pghl-mediated CQ uptake was observed [26]. Taken together these data suggest that overexpression of wild-type Pghl in CHO cells leads to increased acidification of lysosomes the main accumulation site of CQ. Most importantly, direct evidence for a Pghl-mediated acidification of lyso- somes was obtained by probing the lysosomal pH of Pghl-expressing CHO cells by FITC-dextran la- belling. These experiments revealed a significant decrease in lysosomal pH in cells that express Pghl lacking the CQR associated pfmdrl alleles (Fig. 1). Assuming the following relationship [ C Q ] i / [ C Q ] e =

( [ H + ] i / [ H + ] e ) 2 [40], a drop from pH 5.2 to pH 4.8 would result in a [ C Q ] i l / [ C Q ] i 2 = 6.3. Although it is not known what proportion of the intracellular CQ is accumulated in the lysosomes of transfectants and controls, it is likely that the magnitude of the pH decrease is sufficient to explain the increased accu- mulation of CQ. Finally, CQR-associated mutant Pghl did not reveal increased lysosomal acidification indicating the specificity of the observation.

The development of the CQR phenotype in P. falciparum is associated with lower steady-state concentrations of CQ in resistant parasites [11,12,17,21,41]. CQ is a diprotic weak base and therefore an elevated vacuolar pH in CQR parasites may at least in part explain this decrease in CQ accumulation [12,17,21]. An important finding of our previous work was that Pghl carrying the amino acid substitutions S1034C and N1042D does not confer increased CQ sensitivity and increased CQ accumu- lation (Fig. 4) [26]. We have extended these observa- tions and shown here that mutant Pghl does not exhibit an enhanced steady-state lysosomal pH (Fig. 1). This absence of an increase in lysosomal acidifi- cation explains the phenotypic difference between CHO cells expressing wild-type and mutant Pghl. Translated to the situation in P. falciparum para- sites, our results suggest that CQR parasites express- ing mutant Pghl such as isolate 7G8 [16] may have an elevated vacuolar pH as compared to parasites that express a Pghl lacking specific amino acid substitutions at position 1034 or 1042. Suggestive evidence that specific Pghl mutations can result in altered intracellular pH values is provided by the observation that CQR parasites carrying the N86Y CQR associated pfmdrl allele are more sensitive to bafilomycin A1 than CQS parasites [12,16]. This has been interpreted as evidence for an increased vacuo- lar pH due to a weakened vacuolar [H+]ATPase [12]. Alternatively, increased bafilomycin A1 sensitivity could be the result of a defective modulator of lysosomal pH. Mutations in both pH gradient gener- ating and pH gradient modulating proteins can result in an increased vacuolar pH leading to lowered accumulation levels of CQ (see below).

It was long assumed that the mechanism of para- site killing by CQ was the elevation of the vacuolar pH. However, if one assumes that the vacuolar pH is increased in CQR parasites, the digestive vacuole of

Page 9: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

H.H.G. van Es et al. / Molecular and Biochemical Parasitology 68 (1994) 209-219 217

CQR parasites would be alkalinized easier than the vacuole of CQS parasites. Hence, CQR parasites would be more sensitive and not more resistant to CQ [12]. It is now known that an important target of CQ is a heme polymerase, a vacuolar enzyme which is presumably involved in the detoxification of heme liberated during the proteolytic degradation of in- gested hemoglobin [20,24]. Thus, CQ inhibition of heme polymerase is most likely at the basis of the CQR phenotype in malaria parasites. If inhibition of the heme polymerase is indeed the basis of CQ toxicity then differences in pH of the parasite's food vacuole may very well explain the CQR phenotype. Assuming that the observations in p f m d r l trans- fected CHO cells parallel those in the parasite one has to conclude that the increase in the lysosomal CQ concentration due to Pghl-mediated pH decrease inhibits an essential lysosomal component, e.g., acid hydrolases such as a-galactosidase [26,42]. This conclusion has important implications for the inter- pretation of Pghl-mediated drug susceptibility. In- creased susceptibility to weak bases such as CQ, quinine or primaquine will only be detected if a specific target for these drugs is present in the lysosomes. If the specific target is located in the cytoplasm Pghl will confer resistance. Likewise, if no specific target is present within CHO cells, i.e., cytotoxicity is mediated by pH changes only, no striking difference in drug susceptibility between Pghl-expressing and non-expressing cells is ex- pected.

The above data give rise to an important question: How does Pghl mediate or regulate acidification of acidic organelles? We propose that Pghl is an ATP dependent or activated chloride channel which regu- lates chloride permeability of the lysosomal or vac- uolar membrane. Altered C1- permeability will change the relative proportions of A~ and ApH generated by the vacuolar [H÷]ATPase. As would be expected for a charge counterbalancing anion this should facilitate [H÷]ATPase-mediated acidification with a concomitant decrease in A~. Regulation of such a chloride channel would permit the cell to directly regulate the internal pH of organelles such as the malaria parasite's digestive vacuole or mam- malian lysosomes and endosomes. Thus, via pH modulation, vacuolar CI-channels could control critical cellular processes including degradation and

intracellular transport of ingested macromolecules. That Pghl might function as a CI- channel is sup- ported by the observation that CFFR, a well investi- gated ABC-transporter [4], is a chloride channel and by the suggestion that the human P-glycoprotein 1 encoded by M D R 1 is bifunctional and can act as a drug transporter as well as a volume-regulated chlo- ride channel [43,44]. On the other hand, CFFR is only distantly related to Pghl and the C1 channel activity of the MDR1 gene product is controversial [45,46]. Alternatively, it is possible that Pghl is involved in the regulation of the lysosomal H ÷ pump. Such an activation may be analogous to the activation of the S L G T 1 Na/glucose transporter by the membrane associated protein RS1 [47]. The es- tablishment of a cell-free transfectant and control vesicle system will allow to address these and simi- lar hypotheses experimentally.

Acknowledgements

We thank Irving Wainer, Hiltrud Fieger, Sami Abdullah and Alex Faraci for their help with the enantiomer purification and Anne Miller for techni- cal assistance. We greatly appreciate numerous dis- cussions with Dr Ronald Oude Eiferink, AMC, Ams- terdam, the Netherlands. This work was supported by a grant from the Medical Research Council of Canada to ES. ES is a scholar of the Medical Re- search Council of Canada.

References

[1] Higgins, C.F. (1992) ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8, 67-113.

[2] Shepherd, J.C., Schumacher, T.N., Ashton-Rickardt, P.G., Imaeda, S., Ploegh, H.L., Janeway, C.A., Jr. and Tonegawa, S. (1993) TAPl-dependent peptide translocation in vitro is ATP dependent and peptide selective. Cell 74, 577-584.

[3] Neefjes, J.J., Momburg, F. and Hammerling, G.J. (1993) Selective and ATP-dependent translocation of peptides by the MHC-encoded transporter. Science 261,769-771.

[4] Gregory, R.J., Cheng, S.H., Rich, D.P., Marshall, J., Paul, S., Hehir, K., Ostedgaard, L., Klinger, K.W., Welsh, M.J. and Smith, A.E. (1990) Expression and characterization of the cystic fibrosis transmembrane conductance regulator. Nature 347, 382-386.

[5] Smit, J.J.M., Schinkel, A.H., Oude Elferink, R.P.J., Groen,

Page 10: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

218 H.H.G. uan Es et al. /Molecular and Biochemical Parasitology 68 (1994) 209-219

A.K., Wagenaar, E., van Deemter, L., Mol, C.A.A.M., Otten- hoff, R., van der Lugt, N.M.T., van Roon, M.A., van der Valk, M.A., Offenhaus, G.J.A., Berns, A.J.M. and Borst, P. (1993) Homozygous disruption of the murine MDR2 P-gly- coprotein gene leads to a complete absence of phospholipid from lipid and to liver disease. Cell 75, 451-462.

[6] Ortiz, D.F., Kreppel, L., Speiser, D.M., Scheel, G., McDon- ald, G. and Ow, D.W. (1992) Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar membrane transporter. EMBO J. 11, 3491-3499.

[7] Foote, S.J., Thompson, J.K., Cowman, A.F. and Kemp, D.J. (1989) Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum. Cell 57, 921-930.

[8] Cowman, A.F., Karcz, S., Galatis, D. and Culvenor, J.G. (1991) A P-glycoprotein homologue of Plasmodium falci- parum is localized on the digestive vacuole. J. Cell. Biol. 113, 1033-1042.

[9] Goldberg, D.E. and Slater, A.F.G. (1992) The pathway of hemoglobin degradation in malaria parasites. Parasitol. To- day 8, 280-283.

[10] Yayon, A., Cabantchik, Z.I. and Ginsburg, H. (1984) Identi- fication of the acidic compartment of Plasmodium falci- parum-infected human erythrocytes as the target of the anti- malarial drug chloroquine. EMBO J. 3, 2695-2700.

[11] Krogstad, D.J., Gluzman, I.Y., Kyle, D.E., Oduola, A.M., Martin, S.K., Milhous, W.K. and Schlesinger, P.H. (1987) Efflux of chloroquine from Plasmodium falciparum: mecha- nism of chloroquine resistance. Science 238, 1283-1285.

[12] Bray, P.G., Howells, R.E. and Ward, S.A. (1992) Vacuolar acidification and chloroquine sensitivity in Plasmodium fal- ciparum. Biochem. Pharmacol. 43, 1219-1227.

[13] Martin, S.K., Oduola, A.M. and Milhous, W.K. (1987) Re- versal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235, 899-901.

[14] Kyle, D.E., Oduola, A.M., Martin, S.K. and Milhous, W.K. (1990) Plasmodium falciparum: modulation by calcium an- tagonists of resistance to chloroquine, desethylchloroquine, quinine, and quinidine in vitro. Trans. R. Soc. Trop. Med. Hyg. 84, 474-478.

[15] Schinkel, A.H. and Borst, P. (1991) Multidrug resistance mediated by P-glycoproteins. Semin. Cancer. Biol. 2, 213- 226.

[16] Foote, S.J., Kyle, D.E., Martin, R.K., Oduola, A.M., Forsyth, K., Kemp, D.J. and Cowman, A.F. (1990) Several alleles of the multidrug-resistance gene are closely linked to chloro- quine resistance in Plasmodium falciparum Nature 345, 255-258.

[17] Bray, P.G., Howells, R.E., Ritchie, G.Y. and Ward, S.A. (1992) Rapid chloroquine efflux phenotype in both chloro- quine-sensitive and chloroquine-resistant Plasmodium falci- parum. Biochem. Pharmacol. 44, 1317-1324.

[18] Wilson, C.M., Volkman, S.K., Thaithong, S., Martin, R.K., Kyle, D.E., Milhous, W.K. and Wirth, D.F. (1993) Amplifi- cation of pfmdrl associated with mefloquine and halofantrine resistance in Plasmodium falciparum from Thailand. Mol. Biochem. Parasitol. 57, 151-160.

[19] Awad-El-Kariem, F.M., Miles, M.A. and Warhurst, D.C. (1992) Chloroquine-resistant Plasmodium falciparum iso- lates from the Sudan lack two mutations in the pfmdrl gene thought to be associated with chloroquine resistance. Trans. R. Soc. Trop. Med. Hyg. 86, 587-589.

[20] Chou, A.C. and Fitch, C.D. (1992) Heme polymerase: Modu- lation by chloroquine treatment of a rodent malaria. Life Sci. 51, 2073-2078.

[21] Ginsburg, H. and Stein, W.D. (1991) Kinetic modelling of chloroquine uptake by malaria-infected erythrocytes. Assess- ment of the factors that may determine drug resistance. Biochem. Pharmacol. 41, 1463-1470.

[22] Geary, T.G., Jensen, J.B. and Ginsburg, H. (1986) Uptake of [3H]chloroquine by drug-sensitive and -resistant strains of the human malaria parasite Plasmodium falciparum. Biochem. Pharmacol. 35, 3805-3812.

[23] Ginsburg, H. (1990) Antimalarial drugs: Is the lysoso- motropic hypothesis still valid. Parasitol. Today 6, 334-337.

[24] Slater, A.F. and Cerami, A. (1992) Inhibition by chloroquine of a novel haem polymerase enzyme activity in malaria trophozoites. Nature 355, 167-169.

[25] Karcz, S.R., Herrmann, V.R. and Cowman, A.F. (1993) Cloning and characterization of a vacuolar ATPase A subunit homologue from Plasmodium falciparum. Mol. Biochem. Parasitol. 58, 333-344.

[26] van Es, H.H.G., Karcz, S., Chu, F., Cowman, A., Vidal, S., Gros, P. and Schurr, E. (1994) Expression of the plasmodial pfmdrl gene in mammalian cells is associated with increased susceptibility to chloroquine. Mol. Cell. Biol. 14, 2419-2428.

[27] Ohkuma, S. and Poole, B. (1978) Fluorescence probe mea- surements of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. U.S.A. 75, 3327-3331.

[28] Iredale, J. and Wainer, I.W. (1992) Determination of hy- droxychloroquine and its major metabolites in plasma using sequential achiral-chiral high-performance liquid chromatog- raphy. J. Chromatogr. 573, 253-258.

[29] Cain, C.C. and Murphy, R.F. (1986) Growth inhibition of 3T3 fibroblasts by lysosomotropic amines: correlation with effects on intravesicular pH but not vacuolation. J. Cell. Physiol. 129, 65-70.

[30] Yoshimori, T., Yamamoto, A., Moriyama, Y., Futai, M. and Tashiro, Y. (1991) Bafilomycin A1, a specific inhibitor of vacuolar-type H( + )-ATPase, inhibits acidification and pro- tein degradation in lysosomes of cultured cells. J. Biol. Chem. 266, 17707-17712.

[31] Bowman, E.J., Siebers, A. and Altendorf, K. (1988) Bafilomycins: A class of inhibitors of membrane ATPases from microorganisms, animal cells and plant cells. Proc. Natl. Acad. Sci. USA 85, 7972-7976.

[32] Wellems, T.E., Panton, L.J., Gluzman, I.Y., do Rosario, V.E., Gwadz, R.W., Walker-Jonah, A. and Krogstad, D.J. (1990) Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature 345, 253-255.

[33] Ferrari, V. and Cutler, D.J. (1991) Simulation of kinetic data on the influx and effiux of chloroquine by erythrocytes infected with Plasmodium falciparum. Evidence for a drug-

Page 11: Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene

H.H.G. van Es et al. / Molecular and Biochemical Parasitology 68 (1994) 209-219 219

importer in chloroquine-sensitive strains. Biochem. Pharma- col. 42, S167-S179.

[34] Herwaldt, B.L., Schlesinger, P.H. and Krogstad, D.J. (1990) Accumulation of chloroquine by membrane preparations from Plasmodium falciparum. Mol. Biochem. Parasitol. 42, 257- 267.

[35] Wellems, T.E., Walker-Jonah, A. and Panton, L.J. (1991) Genetic mapping of the chloroquine-resistance locus on Plasmodium falciparum chromosome 7. Proc. Natl. Acad. Sci. U.S.A. 88, 3382-3386.

[36] Ekong, R.M., Robson, K.J.H., Baker, D.A. and Warhurst, D.C. (1993) Transcripts of the multidrug resistance genes in chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. Parasitology 106, 107-115.

[37] Ott, R.J. and Giacomini, K.M. (1993) Stereoselective trans- port of drugs across epithelia. In: Drug Stereochemistry. Analytical Methods and Pharmacology (Wainer, I.W., ed.), pp. 281-314. Marcel Dekker, New York.

[38] Gros, P., Talbot, F., Tang Wai, D., Bibi, E. and Kaback, H.R. (1992) Lipophilic cations: A group of model substrates for the multidrug-resistance transporter. Biochemistry 31, 1992-1998.

[39] Mancini, G.M., de Jonge, H.R., Galjaard, H. and Verheijen, F.W. (1989) Characterization of a proton-driven carrier for sialic acid in the lysosomal membrane. Evidence for a group-specific transport system for acidic monosaccharides. J. Biol. Chem. 264, 15247-15254.

[40] Moreau, S. (1989) La chloroquine: m6canisme d'action anti- malarique et resistance. Mddecine/Sciences 9, 729-735.

[41] Krogstad, D.J., Gluzman, I.Y., Herwaldt, B.L., Schlesinger, P.H. and Wellems, T.E. (1992) Energy dependence of chloroquine accumulation and chloroquine efflux in Plas- modium falciparum. Biochem. Pharmacol. 43, 57-62.

[42] de Groot, P.G., Oude Elferink, R.P.J., Hollemans, M., Strij- land, A., Westerveld, A., Meera Khan, P. and Trager, J.M. (1981) Inactivation by chloroquine of alfa-galactosidase in cultured human skin fibroblasts. Exp. Cell Res. 136, 327-333.

[43] Gill, D.R., Hyde, S.C., Higgins, C.F., Valverde, M.A., Mintenig, G.M. and Sepfilveda, F.V. (1992) Separation of drug transport and chloride channel functions of the human multidrug resistance P-glycoprotein. Cell 71, 23-32.

[44] Valverde, M.A., Diaz, M., Sepulveda, F.V., Gill, D.R., Hyde, S.C. and Higgins, C.F. (1992) Volume-regulated chloride channels associated with the human multidrug-resistance P- glycoprotein. Nature 355, 830-833.

[45] Rasola, A., Galietta, L.J.V., Gruenert, D.C. and Romeo, G. (1994) Volume-sensitive chloride currents in four epithelial cell lines are not directly correlated to the expression of the MDR-1 gene. J. Biol. Chem. 269, 1432-1436.

[46] Altenberg, G.A., Vanoye, C.G., Han, E.S., Deitmer, J.W. and Reuss, L. (1994) Relationship between rhodamine 123 trans- port, cell volume, and ion-channel function of P-glycopro- tein. J. Biol. Chem. 269, 7145-7149.

[47] Veyhl, M., Spangenberg, J., Puschel, B., Poppe, R., Dekel, C., Fritzsch, G., Haase, W. and Koepsell, H. (1993) Cloning of a membrane-associated protein which modifies activity and properties of the Na( + )-D-glucose cotransporter. J. Biol. Chem. 268, 25041-25053.