Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, C13 and PC13
Human erythrocyte peripheral type benzodiazepine receptor/voltage-dependent anion channels are up-regulated by Plasmodium falciparum
G. Bouyer1,2, A. Cueff1,2, S. Egee1,2, J. Kmiecik1,2, Y. Maksimova3, E. Glogowska1,2, P. Gallagher3, S. Thomas1,2
1. UMR7150, CNRS, Roscoff, France. 2. UMR7150, UPMC, ROSCOFF, France. 3. Departments of Pediatrics and Genetics, Yale University School of Medicine, New Haven, Connecticut, United States.
Plasmodium falciparum relies on anion channel activity in the erythrocyte membrane to ensure the transport of nutrients and waste products associated with its development after invasion. The molecular identity of these “New Permeability Pathways (NPP)” described almost thirty years ago (1), is unknown, but their currents correspond to up-regulation of endogenous channels displaying complex gating and kinetics (2-3). In a previous work (4), we had shown that a maxi-anion channel was present in the human red cell membrane. We now demonstrate that components of the Peripheral-type Benzodiazepine Receptor (PBR) are present and functional in the human erythrocyte membrane, and suggest their implication in the increased permeability of infected cells. The PBR, originally described in mitochondrial membranes, consists of at least three components: Voltage-Dependant Anion Channel (VDAC), Translocator protein (TSPO), and Adenine Nucleotide Transporter (ANT). First, using RT-PCR, we showed that transcripts for the three subunits can be found in the erythroid cell line and CD34+ cells, compared to Ramos cells as a standard. We also demonstrated by western blots, mass spectrometry and immunofluorescence staining that the proteins are present in the mature human erythrocyte membrane (venous blood obtained from healthy volunteers, with written informed consent; all experiments done at least three times). Secondly we tested the hypothesis whereas this PBR could be the molecular support of the NPP activity described in P. falciparum-infected erythrocytes. PBR are characterised by nanomolar affinity for the ligands PK11195>Ro5-4864>Diazepam (5). The three ligands were shown to be more potent in inhibiting P. falciparum growth in vitro than NPPB, a classical NPP inhibitor. IC50 were 13.2, 62.8 and 80.5 µM for PK11195, Ro5-4864 and diazepam, respectively, compared to 97.6µM for NPPB (n=3). These ligands also reduced membrane transport of infected erythrocytes, as seen by isosmotic sorbitol haemolysis (characteristic of NPP activity). Analysis showed that the half-times of lysis (t1/2) was significantly increased (P<0.001, student’s t-test) when concentration reached 50 μM, 1 μM and 10μM for PK11195, Ro5-4864 and Diazepam, respectively (n=3). Using the whole-cell configuration of the patch clamp technique, we also showed (n=6 for each condition) that the three ligands reduced the conductance in P. falciparum-infected erythrocytes (representative traces are shown in figure 1). These data support the hypothesis that the dormant PBR mediates the band 3-independent anion conductance in normal erythrocytes, and, after up-regulation by P. falciparum, becomes the “New Permeability Pathways” in infected erythrocytes. These channels are obvious targets in the fight against malaria. All these results are included in an article currently in press in Blood journal.
Where applicable, experiments conform with Society ethical requirements