Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo-4
Version of Record online: 1 JUN 2014
© 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Journal of Cellular and Molecular Medicine
Volume 18, Issue 9, pages 1851–1862, September 2014
How to Cite
Friedrich, O., Reiling, S.J., Wunderlich, J. and Rohrbach, P. (2014), Assessment of Plasmodium falciparum PfMDR1 transport rates using Fluo-4. Journal of Cellular and Molecular Medicine, 18: 1851–1862. doi: 10.1111/jcmm.12313
- Issue online: 10 SEP 2014
- Version of Record online: 1 JUN 2014
- Manuscript Accepted: 31 MAR 2014
- Manuscript Received: 25 NOV 2013
- German Academic Exchange Service. Grant Number: 50748369
- Québec Ministère des Relations Internationales
- Natural Sciences and Engineering Research Council
- Bayerische Forschungsallianz
- Canada Foundation for Innovation (CFI) Leaders Opportunity Fund
- Erlangen Graduate School of Advanced Optical Technologies
- Plasmodium falciparum ;
- transport kinetics;
- live-cell imaging;
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.