Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo-4

Mutations in the multidrug resistance transporter of Plasmodium falciparum PfMDR1 have been implicated to play a significant role in the emergence of worldwide drug resistance, yet the molecular and biochemical mechanisms of this transporter are not well understood. Although it is generally accepted that drug resistance in P. falciparum is partly associated with PfMDR1 transport activity situated in the membrane of the digestive vacuole, direct estimates of the pump rate of this transport process in the natural environment of the intact host–parasite system have never been analysed. The fluorochrome Fluo-4 is a well-documented surrogate substrate of PfMDR1 and has been found to accumulate by actively being transported into the digestive vacuole of several parasitic strains. In the present study, we designed an approach to use Fluo-4 fluorescence uptake as a measure of compartmental Fluo-4 concentration accumulation in the different compartments of the host–parasite system. We performed a ‘reverse Fluo-4 imaging' approach to relate fluorescence intensity to changes in dye concentration rather than Ca2+ fluctuations and were able to calculate the overall rate of transport for PfMDR1 in Dd2 parasites. With this assay, we provide a powerful method to selectively measure the effect of PfMDR1 mutations on substrate transport kinetics. This will be of high significance for future compound screening to test for new drugs in resistant P. falciparum strains.


Introduction
Malaria remains a major health problem in many parts of the world, particularly sub-Saharan Africa, where~216 million cases of infection and 665,000 to 1.2 million deaths occur annually [1,2]. The emergence and spread of multidrug-resistant Plasmodium falciparum parasites have severely compromised the ongoing global efforts to control the disease [3]. P-glycoprotein transporters are often implicated in disease aetiology and treatment. Investigations into P. falciparum resistance have included pfmdr1, a parasite homologue of the mammalian multidrug resistance ATP binding cassette (ABC) transporter family. Pfmdr1 encodes a 162 kD protein that consists of two homologous parts, each comprising a cytosolic nucleotide-binding domain (NBD) and a substrate-binding transmembrane domain (TMD) with 12 putative transmembrane regions.
In the malaria parasite, PfMDR1 has been localized to the membrane of the parasite's digestive vacuole (DV) [4], an acidic lysosome-like organelle [5], and has been shown to transport solutes into the DV [6]. If similar to MDR1 transporters in the plasma membrane of mammalian cells, PfMDR1 is anticipated to bind its substrates in the cytosolic membrane leaflet of the DV membrane and flip them to the inner DV leaflet at the expense of ATP hydrolysis.
PfMDR1, as all MDR1 transporters, has broad substrate specificity, enabling the transport of a diverse array of substances. In the past, fluorochromes have been used in a wide range of assays to assess P-gp function [7]. Recently, we showed that the fluorochrome Fluo-4 is a substrate for PfMDR1. Fluo-4 is commonly used as a non-ratiometric Ca 2+ indicator in eukaryotic cells but has also been used to evaluate multidrug transporter activity in lymphocytes [8]. In P. falciparum, Fluo-4 provided evidence for *Correspondence to: P. Rohrbach, active transport of the dye into the parasite's DV [6]. Furthermore, the mutant pfmdr1 gene (Y86, Y184, S1034, N1042, D1246) encodes for a protein that is more effective in transporting Fluo-4 from the cytosol of the parasite into the DV than the wild-type PfMDR1 protein (N86, F184, S1034, D1042, D1246) [6]. Although these studies showed that, under steady-state conditions, the accumulated staining pattern of Fluo-4 fluorescence in the DV was a marker to differentiate between drug-sensitive versus -resistant strains [6], the kinetics of this transport process reflecting the elementary mode of action of this drug resistance transporter remained elusive.
Understanding the bioenergetics of PfMDR1 sheds light on general mechanisms of action for this superfamily of transporters. Unlike artificial systems, such as PfMDR1 embedded in artificial liposomes, it is desirable to study transport rates in the natural environment of the transporter, i.e. within the DV of the intact P. falciparum-infected erythrocyte and to have tools at hand to differentiate between pump rates and diffusional uptake. Therefore, the aim of the present study was to investigate the transport kinetics of PfMDR1 in the host-parasite system using the Fluo-4 fluorochrome and live-cell imaging. We describe a detailed investigation of the transporter in relation to Fluo-4 solute uptake applied in all compartments of the host-parasite system (Fig. 1). This is the first estimate of overall transport rates of PfMDR1 in the intact host-parasite system. Using a ligand-binding model, we were able to estimate a maximum overall pump rate of 50,000 Fluo-4 molecules/min.-or 820/sec.-for the complete set of PfMDR1 molecules found on the DV membrane of the Dd2 parasite.

Cultivation of P. falciparum parasites
Plasmodium falciparum blood stage parasites (HB3, Dd2) were maintained in culture using a modified protocol of Trager and Jensen [9]. Parasites were propagated using human A + erythrocytes in complete RPMI medium supplemented with 0.5% AlbuMAX II (Life Technologies Inc., Burlington, ON, Canada) at 37°C in an atmosphere of 92% N 2 , 3% O 2 , 5% CO 2 . All experiments were carried out using trophozoite stage parasites (28-36 hrs post invasion).  [10] are of no concern here. Dye-loaded parasites were settled onto poly-L-lysine-coated coverslips in a micro-perfusion chamber as described in [11]. Unbound parasites and remaining dye were washed away by perfusion with Ringer's solution.

Image analysis
P. falciparum-infected erythrocytes were readily identified by eye. Using the Zen software (Carl Zeiss), a region of interest within the host-parasite system, e.g. the DV, the cytoplasm of the parasite, the cytosol of the infected RBC or the uninfected RBC, was defined. The average pixel intensity of the region of interest was calculated. The measured fluorescence was further analysed using Excel (Microsoft Canada Corp., Quebec,

pfmdr1 sequence analysis
The full length sequence of pfmdr1 was verified in our laboratory HB3 and Dd2 strains. Parasite strains were grown to ≥5% parasitaemia and DNA extracted using the QIAamp DNA Blood Mini Kit (Qiagen Inc., Toronto, ON, Canada) according to the manufacturer's instructions. The DNA was amplified in overlapping PCR fractions using HotStarTaq DNA polymerase (Qiagen Inc.). To account for the AT-rich nucleotide content in the P. falciparum genome, dNTPs (Invitrogen Canada Inc., Burlington, ON, Canada) were mixed at 75% AT and 25% GC. For PCR optimization, 2 mM MgCl 2 , 300 lM dNTPs and 300 nM primers were used for the reaction. For each reaction mix, a total of 20 ng genomic DNA was used. PCR reactions consisted of an initial activation step of 94°C for 3 min. followed by 35 cycles of 94°C for 60 sec., 49-61°C (adjusted for each primer pair) for 30 sec. and 72°C for 1 min. Primers used for pfmdr1 gene sequencing were: If DNA amplification using pfmdr1.7 primers was unsuccessful, the PCR was repeated with the alternative primers pfmdr1.7.1. Samples were sent for sequencing to Genome Quebec, Canada and analysed using the BioEdit software [14].

Real-time PCR
pfmdr1 gene copy numbers were determined through real-time PCR using fluorescent TaqMan probe-based gene expression. Primer pairs of pfmdr1 [15] and the housekeeping gene seryl-t-rna-synthetase were designed to match in length and nucleotide content of the two amplified gene regions. In addition, TaqMan probes with a FAM or VIC dye on the 5 0 end and a TAMRA quencher on the 3 0 end were added to the reaction mix (Applied Biosystems, USA). The primers used for quantifying copy number were: PF-F: . Samples were prepared in triplicates. For the reaction, the initial activation step was at 94°C for 3 min., followed by 40 cycles of 94°C for 1 min., 60°C for 1 min. and 72°C for 30 sec. Fluorescence was recorded after each elongation step.
Real-time PCR was carried out in a Rotor-Gene RG-3000 (Corbett Research, Toronto, ON, Canada). Copy numbers were determined relative to 3D7, which is known to have only one pfmdr1 gene copy [16,17].

Results
Fluo-4 fluorescence accumulation in P. falciparum-infected erythrocytes In a previous study, we showed that the fluorochrome Fluo-4 is actively transported into the DV of chloroquine-resistant (CQR) Dd2 but not chloroquine-sensitive (CQS) HB3 parasites via the multidrug resistance transporter PfMDR1 [6]. In the present study, we have extended this approach on live cells using Fluo-4 fluorescence detection in P. falciparum-infected erythrocytes as an in situ assay to directly monitor vacuolar PfMDR1 transport and establish the overall pump rate of this transporter in Dd2 parasites. This is possible as the increase in Fluo-4 fluorescence in the DV is related to the dye accumulation in this compartment via diffusion and PfMDR1 transport activity (Fig. 1). PfMDR1 activity can thus be dissected out  comparing group data from HB3 strains (pure diffusion model) and Dd2 strains (diffusion and active pump model).
To determine live in situ Fluo-4 uptake rates in the intact hostparasite system, it is essential to compare the global fluorescence increase representing the Fluo-4 uptake within CQS and CQR parasites to verify for possible differences in global uptake kinetics (i.e. all compartments merged). Evaluation of total global Fluo-4 fluorescence signals within the intact HB3 or Dd2 P. falciparum-infected erythrocyte (comprising all compartments of the host-parasite system, including the erythrocyte cytosol, parasite cytoplasm and DV) revealed no significant difference in fluorochrome uptake kinetics within the 40 min. time interval investigated (Fig. 2A). The temporal profiles of the integrated fluorescence intensity show a complete overlap for both strains, suggesting a linear increase in global Fluo-4 accumulation. However, when the fluorescence signals within the respective single compartments of the host-parasite system were investigated (i.e. erythrocyte cytosol, parasite cytoplasm, DV), Fluo-4 accumulation varied substantially among the compartments of the two strains. While HB3 parasites generally showed a uniform Fluo-4 accumulation throughout the whole parasite (difference not significant), Dd2 parasites displayed an increased accumulation of Fluo-4 signal in the DV (Fig. 2B). This prompted us to focus on the time resolved quantification of Fluo-4 fluorescence in each separate compartment.
Our previous work showed that the accumulation of Fluo-4 in the DV of Dd2 parasites is mainly due to the pump action of PfMDR1, which is modulated through mutations in this transporter [6]. HB3 parasites do not transport Fluo-4 into the DV and take up this dye through passive diffusion (Fig. 1). To verify mutations that facilitate Fluo-4 transport, we sequenced the complete pfmdr1 gene for both HB3 and Dd2 parasites cultured in our laboratory in addition to determining the gene copy numbers of these strains. We confirmed that only three PfMDR1 polymorphisms at amino acid positions 86, 184 and 1042 differed between the two parasites (Table 1). Interestingly, the PfMDR1 amino acid sequence of Dd2 revealed both tyrosine (Y) and phenylalanine (F) at position 86. A serial dilution of this strain was carried out and re-sequencing confirmed the Y/F mutation. Pfmdr1 copy numbers for HB3 and Dd2 parasites were verified and calculated to be 1 and 2, respectively. Because of amino acid alterations seen in our Dd2 strain, an MR4obtained Dd2 strain (MRA-150, MR4, ATCC â , Manassas, VA, USA) was sequenced to confirm the amino acid variation seen at position 86. The pfmdr1 copy number of this MR4 strain was found to be 3 (Table 1). For this study, we used the Dd2 strain containing two copies of pfmdr1.
To deduce kinetics properties for PfMDR1, one must follow the time course of Fluo-4 fluorescence (F Fluo-4 ) accumulation in the hostparasite system. To evaluate F Fluo-4 increases in the various host-parasite compartments over time, we measured F Fluo-4 within this system over 3 hrs using five different external Fluo-4 AM concentrations in the bulk solution (0.1-15 lM). For both HB3-and Dd2-intact multicompartment systems, the time course of F/F(0) (normalized F Fluo-4 ) during dye loading was quantified for the iRBC cytosol, the parasite cytoplasm and the parasite's DV (Fig. 3). The normalized fluorescence F(t) = F/F(0) of Fluo-4 dye loading was found to be comparable in the iRBC and parasite cytoplasm compartments of HB3 and Dd2 parasites but differed significantly for the DV.

Apparent K d of Fluo-4
As Fluo-4 only fluoresces when bound to free Ca 2+ , we sought out to quantify the K d for the reaction of this dye in each compartment of the parasite. The increase in fluorescence intensity with increasing [Ca 2+ ] was used in an in situ calibration procedure to obtain the apparent steady-state K d of the dye. To calibrate F Fluo-4 to [Ca 2+ ] free in each compartment, the normalized steady-state fluorescence intensity F/F(0 Ca 2+ ) (normalized to base intensities at diminished Ca 2+ levels of pCa~9) was measured (n > 20 parasites). Figure 4 shows the calibration results in the host erythrocyte compartments of uninfected (RBC) and HB3-or Dd2-infected erythrocytes (iRBC) as well as the parasitic compartments. Using the fit-derived K d values, the apparent steady-state Ca 2+ levels for each compartment were determined from resting F Fluo-4 values as given in Table 2. It is important to note that the right-shifted pCa-Fluo-4 plot for the DV of Dd2 parasites in Figure 4 suggests a weaker binding affinity of Fluo-4 to Ca 2+ in this compartment when compared to HB3 parasites.

Fluo-4 concentration accumulation in the intact host-parasite system
The greater Fluo-4 fluorescence values seen for Dd2 parasites would suggest that the DV of this strain contains high concentrations of Ca 2+ (Table 2). This is misleading and was proven to be incorrect by using more reliable ratiometric Fura-Red recordings, which delivered actual steady-state Ca 2+ concentrations well below the measured Fluo-4 values (Table 2) [12]. As the quantified [Ca 2+ ] free represent steady-state values and PfMDR1 actively pumps Fluo-4 into the DV of Dd2 parasites, the pump rate of this transporter cannot be deduced from these values. Furthermore, despite the fact that the bulk concentration of dye is fixed under our conditions, each compartment does not necessarily equilibrate to this dye-level value in the steady-state, in particular not the DV where dye is actively concentrated. Therefore, to deduce the kinetics properties of PfMDR1 on the DV membranes of Dd2 parasites, the measured time course of Fluo-4 fluorescence uptake must be converted into an absolute dye concentration using an equation that also takes into account the differences in pH values between the parasite's cytosol (pH 7.2) and DV (pH 5.2).
The fluorescence-Ca 2+ projections from the fluorescence-[Ca 2+ ] free -[Fluo-4] tot landscape of the in vitro calibration is shown in Figure 5A. Fluorescence projections for selected total  values at pH 5.2 and 7.2 from these landscapes (both absolute fluorescence values and normalized to background values are shown) are depicted in the left panels of Figure 5A. Sigmoidal fits were used to extract the K d values of the Fluo-4-Ca 2+ buffer reaction for all  tot investigated at each pH (Fig. 5A). As the K d values do not depend on total Fluo-4 concentration in a closed system (the equilibrium at any given fixed Ca 2+ concentrations, Eq. (1), just shifts towards larger fluorescence values for larger total dye concentrations, Fig. 5A), the experimental K d values were averaged together for all [Ca 2+ ] at pH 5.2 and 7.2, respectively, as they were not systematically different within each pH value (data not shown). The evaluated K d values for the Ca 2+ -Fluo-4 system indicate a roughly 40 times lower affinity for Fluo-4 Ca 2+ binding at pH 5.2 (Fig. 5B).
Using the known steady-state free Ca 2+ levels ([Ca 2+ ] free ) from past Fura-Red studies ( Table 2) and the K d values at the pH values of interest (5.2 for DV, 7.2 for parasite cytoplasm and iRBC cytosol), Eq. (1) was used to construct a linear calibration curve relating Fig. 4 Normalized pCa-Fluo-4 steady-state fluorescence relations. In situ calibration relationships were applied to obtain apparent K d values in the different compartments of HB3-and Dd2-infected erythrocytes. Steady-state Fluo-4 fluorescence intensities are given for different clamped pCa values in ionomycin-permeabilized HB3-or Dd2-infected erythrocytes (iRBC) or non-infected erythrocytes (RBC). Within the parasites, the cytoplasmic and DV compartments were evaluated. Fluorescence values were normalized to the basal fluorescence intensity at pCa 9 (essentially no Ca 2+ present) and the apparent K d values extracted from the sigmoidal relationships. External Fluo-4 AM concentration was 5 lM. The iRBC represents a low-affinity compartment compared to RBC cytosol, most likely due to the drain of Ca 2+ into the parasitic compartments. From the reconstructed calibration curves, the apparent Fluo-4 fluorescence derived compartmental Ca 2+ concentrations were obtained (see Table 2).   (Fig. 5C). With this calibration, the fluorescence uptake results from Figure 2 were converted to absolute total compartmental [Fluo-4] concentration ([Fluo-4] compartment ) curves (Fig. 6).

Fluo-4 uptake kinetics
Using the kinetics time course data (Fig. 6), we sought to construct a dose-response curve for the relationship of external total [Fluo-4] bulk and internal total [Fluo-4] compartment for each compartment using the steady-state values (Fig. 7A). All compartments, except the DV of Dd2, demonstrate a linear relationship. The 'Dd2 bulk Fluo-4 input' to 'vacuolar Fluo-4 concentration' relationship (Bulk-[Fluo-4] À [Fluo-4] DV ) fitted well with a ligand-binding model containing a one-site saturation plus a non-specific binding site of the equation corresponding  values from both compartments at corresponding time-points from Figure 6 were plotted (Fig. 7B). For HB3 parasites, the vacuolar-cytoplasmic  relationship is linear for all bulk concentrations used and is compatible with a passive accumulation of Fluo-4 in the HB3 DV (diffusion model). For Dd2, cytoplasmic  reached greater values compared to HB3 cytoplasm. Moreover, for a given cut-off value for cytoplasmic  in Dd2 parasites, vacuolar [Fluo-4] bent off to reach a several-fold larger concentration because of active accumulation of Fluo-4 via PfMDR1 (Fig. 7B).
To assess the in situ uptake rates of Fluo-4, the time course curves of Figure 6 were differentiated in time for the Dd2 and HB3 compartments (Fig. 7C). As Dd2 curves comprise the combined contribution of passive and active vacuolar solute transport, the difference in kinetics (Dd2 À HB3) provides the active transport rate of the PfMDR1 pumps, assuming a similar passive distribution in both HB3 and Dd2 parasites (Fig. 7C, Dd2 À HB3). The vacuolar curves describe exponentially decaying PfMDR1 pump rates for Fluo-4 uptake in situ that have concentration-dependent maximum values between 8 and 15 lM/min. for the bulk Fluo-4 concentrations of 1-15 lM tested. From the final vacuolar concentrations (Fig. 6) and the morphometric analysis of the compartmental volumes (Table 3), we can estimate the number of Fluo-4 molecules in the steady-state to be in the order of 3 Mio in the DV of Dd2 parasites with a maximum overall pump rate of~50,000 Fluo-4 molecules/ min. or 820/sec. for all PfMDR1 molecules present on the DV membrane.

Discussion
It is well known that drug resistance in P. falciparum is related to PfMDR1 activity in the membrane of the DV as an ATP-consuming process, however, direct estimates of the pump rate of this transport process in the natural environment of the intact host-parasite system have never been assessed. As previous studies have shown that Fluo-4 is a substrate for PfMDR1 [6,12], we sought to design an approach to use Fluo-4 fluorescence uptake as a measure of compartmental Fluo-4 concentration accumulation in the different compartments of the host-parasite system during dye loading. We performed a 'reverse imaging' approach and successfully calculated the rate of transport for PfMDR1 in Dd2 parasites using a ligand-binding model containing one-site saturation and non-specific binding.
The transport of Fluo-4 is mainly influenced by distinct amino acid residues-and not copy number-of the PfMDR1 transporter [6]. We evaluated pfmdr1 copy numbers for the HB3 and Dd2 parasite strains cultured in our laboratory and found them to have 1 and 2 copies, respectively. To verify for PfMDR1 polymorphisms, we sequenced the complete pfmdr1 gene of HB3 and Dd2 parasites to ensure that no additional mutations were present that may influence substrate transport. We confirmed three single nucleotide polymorphisms in the two strains at positions 86, 184 and 1042. While HB3 parasites contained N86, F184, D1042 polymorphisms, Dd2 parasites had F/Y86, Y184, N1042. Dd2 parasites revealed an additional variation at position 86, in which one of the two copies of pfmdr1 contained the amino acid F86 and the other Y86. PfMDR1 mutations at position 86 have been suggested to play a significant role in multidrug resistance [18][19][20], although numerous studies have also shown that positions 184, 1034, 1042 and 1246 could also be important [15,[21][22][23][24]. Our data propose that polymorphisms at this position are important for the transport of Fluo-4 into the DV of Dd2 parasites. HB3 parasites do not transport Fluo-4 into the DV and seem to only accumulate the dye by passive diffusion.
To better understand the role of PfMDR1 in multidrug resistance, a more comprehensive biochemical characterization (i.e. pump rates and kinetics) of this transporter is required. Rather than expressing PfMDR1 in an artificial expression system, which could introduce artefacts to its mode of action, our approach deduces PfMDR1 pump rates directly from intact host-parasite Fluo-4 fluorescence uptake data. Calculating the overall transport rate of PfMDR1 required the conversion of Fluo-4 uptake fluorescence into absolute Fluo-4 concentrations. For this, several parameters were quantified. First, the apparent steady-state K d was calculated in the respective compartments of live parasites, as these values are profoundly influenced by environmental parameters such as pH, temperature and ionic strength [12]. This must be taken into account when interpreting Fluo-4 fluorescence data in live cells, otherwise the conclusions drawn from the resulting data could be misconstrued. For example, in the past, the DV was suggested to serve as a Ca 2+ store [25]. This conclusion, however, is incorrect as the above mentioned variables were not applied to the measured Fluo-4 fluorescence. When ratiometric Ca 2+ dyes were used, it was shown that the DV is not a Ca 2+ store [12]. Nevertheless, Fluo-4 can be used for live-cell imaging of parasites in another way, as it is a substrate for PfMDR1 and can serve as a valuable asset for monitoring PfMDR1 pump activity through time-lapse recordings of Fluo-4 fluorescence intensities during dye loading protocols. For this approach, steady-state free [Ca 2+ ] were assumed as fixed for each compartment and taken from our previous work, where we used Fura-Red to measure resting Ca 2+ concentrations in Dd2 and HB3 parasites [12].  Figure 6, the parasitic cytoplasm-DV relationship was reconstructed, which represents the driving force for Fluo-4 from the cytoplasm into the DV. For HB3, this process is entirely compatible with diffusion while for Dd2, diffusion would only allow for a limited vacuolar uptake that is vastly exceeded by the active pump. (C) By differentiating the time courses of the uptake fits from Figure 6, Fluo-4 uptake rates are acquired and plotted for the cytoplasm and DV of Dd2 and HB3 parasites, respectively. The plot Dd2-HB3 (far right panel) shows diminished cytoplasmic rates while providing only uptake kinetics via the PfMDR1 pump. and 7.2 in this study. With this information, absolute Fluo-4 concentrations for the various compartments of the host-parasite system were obtained for the first time.
Dose-response curves of internal [Fluo-4] compartment at given bulk Fluo-4 concentrations demonstrate that at 1 lM external Fluo-4, the vacuolar Fluo-4 concentration is already saturated in the steady-state between 900 lM and 1 mM. The resulting uptake curves are virtually the same in the iRBC cytosol and the parasite cytoplasm for both HB3-and Dd2-infected erythrocytes. The major difference lies in the vacuolar uptake, which is greatly increased in the DV of Dd2 parasites and reaches extrapolated steady-state values close to~1 mM Fluo-4 for the larger bulk concentrations of 5 and 15 lM after~200 min. Despite the similar uptake kinetics in the outer compartments, iRBC uptake is nevertheless greater compared to uninfected erythrocytes, which can be explained by the larger drain of Fluo-4 into the parasite compartments of iRBCs.
The vacuolar difference curves of Dd2 À HB3, reflecting the pure PfMDR1 pump behaviour, describe exponentially decaying PfMDR1 pump rates for Fluo-4 uptake in situ with concentrationdependent maximum values between 8 and 15 lM/min. for the tested bulk Fluo-4 concentrations of 1-15 lM. This decline in pump rate under in situ conditions is expected, as ongoing pump activity fills up the DV with Fluo-4 molecules, which in turn slows down further Fluo-4 uptake. From the final vacuolar concentrations and the morphometric analysis of the compartmental volumes (Table 3), we can estimate the number of steady-state Fluo-4 molecules to be in the order of 3 million in the DV of Dd2 parasites with a maximum pump rate of~50,000 Fluo-4 molecules/min.-or 820/sec.-across the DV membrane as a whole. Although it would be good to have the actual pump rate per molecule of PfMDR1, quantitative assessments of the exact expression density of transport molecules have never been obtained. Without knowing the pump rate per molecule of PfMDR1, the conclusions drawn from our current study can only reflect overall pump rate of PfMDR1 in the DV of Dd2 parasites. This must be taken into consideration when comparing different drug-sensitive strains using our method-ology in future studies, as differences in overall pump rates may not only relate to differences in pfmdr1 copy numbers but also to differential PfMDR1 expression density on the DV membrane. We are addressing this issue in future studies using super-resolution microscopy in conjunction with fluorescently tagged PfMDR1.
In conclusion, this is the first direct quantification of in situ overall DV pump rates for PfMDR1 in the intact host-parasite system. We were able to determine the rate of solute transport across the DV membrane of Dd2 parasites with the PfMDR1 mutations F/ Y86, Y184, S1034, N1042, and D1246. This assay has provided us with a powerful tool to measure the effect of PfMDR1 mutations and copy numbers on substrate (Fluo-4) import kinetics. The methodology can be implemented to monitor drug resistance associated with distinct mutations in the transporter and will be used to screen small molecule libraries available in our laboratories. We are now able to identify which PfMDR1 amino acid mutations can more effectively modulate the transport of any given drug or substrate into the DV. In turn, this will allow for a better understanding of the transporter's mode of action and possibly reveal its natural substrates to us.