Limits...
Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo-4.

Friedrich O, Reiling SJ, Wunderlich J, Rohrbach P - J. Cell. Mol. Med. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany; SAOT, Erlangen Graduate School of Advanced Optical Technologies, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.

Show MeSH

Related in: MedlinePlus

Proposed model of Fluo-4 distribution within the compartments of Plasmodium falciparum (Dd2)-infected erythrocytes. Bulk [Fluo-4]-AM is taken up into the erythrocyte cytosol by passive diffusion, where the acetoxymethyl-ester is partially cleaved by esterases, making it membrane impermeant. Uncleaved Fluo-4 AM is being redirected into the parasite's cytoplasm through the PV membrane, where it is cleaved and slowly accumulates in this compartment. In the Dd2 parasite, transport of Fluo-4 into the digestive vacuole occurs both via diffusion (Fluo-4 AM) and by active transport via the PfMDR1 pump, which transports both the AM and the cleaved Fluo-4 dye.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4196660&req=5

fig01: Proposed model of Fluo-4 distribution within the compartments of Plasmodium falciparum (Dd2)-infected erythrocytes. Bulk [Fluo-4]-AM is taken up into the erythrocyte cytosol by passive diffusion, where the acetoxymethyl-ester is partially cleaved by esterases, making it membrane impermeant. Uncleaved Fluo-4 AM is being redirected into the parasite's cytoplasm through the PV membrane, where it is cleaved and slowly accumulates in this compartment. In the Dd2 parasite, transport of Fluo-4 into the digestive vacuole occurs both via diffusion (Fluo-4 AM) and by active transport via the PfMDR1 pump, which transports both the AM and the cleaved Fluo-4 dye.

Mentions: 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.


Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo-4.

Friedrich O, Reiling SJ, Wunderlich J, Rohrbach P - J. Cell. Mol. Med. (2014)

Proposed model of Fluo-4 distribution within the compartments of Plasmodium falciparum (Dd2)-infected erythrocytes. Bulk [Fluo-4]-AM is taken up into the erythrocyte cytosol by passive diffusion, where the acetoxymethyl-ester is partially cleaved by esterases, making it membrane impermeant. Uncleaved Fluo-4 AM is being redirected into the parasite's cytoplasm through the PV membrane, where it is cleaved and slowly accumulates in this compartment. In the Dd2 parasite, transport of Fluo-4 into the digestive vacuole occurs both via diffusion (Fluo-4 AM) and by active transport via the PfMDR1 pump, which transports both the AM and the cleaved Fluo-4 dye.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196660&req=5

fig01: Proposed model of Fluo-4 distribution within the compartments of Plasmodium falciparum (Dd2)-infected erythrocytes. Bulk [Fluo-4]-AM is taken up into the erythrocyte cytosol by passive diffusion, where the acetoxymethyl-ester is partially cleaved by esterases, making it membrane impermeant. Uncleaved Fluo-4 AM is being redirected into the parasite's cytoplasm through the PV membrane, where it is cleaved and slowly accumulates in this compartment. In the Dd2 parasite, transport of Fluo-4 into the digestive vacuole occurs both via diffusion (Fluo-4 AM) and by active transport via the PfMDR1 pump, which transports both the AM and the cleaved Fluo-4 dye.
Mentions: 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.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biotechnology, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany; SAOT, Erlangen Graduate School of Advanced Optical Technologies, Friedrich-Alexander-University Erlangen-Nuremberg, Erlangen, Germany.

Show MeSH
Related in: MedlinePlus