STEPHANE EGEE & SERGE THOMAS


Email: egee@sb-roscoff.fr and thomas@sb-roscoff.fr

Team: “Physiologie comparée des érythrocytes”

Website: http://www.sb-roscoff.fr/

The team ‘Physiologie comparée des érythrocytes’ of the UMR 7150 CNRS-UPMC was created in 1999 in Roscoff Station biologique. The experience of Serge Thomas in the field of comparative respiration physiology since 1977 oriented the research activity towards description at the molecular level of ion channels present in the membrane of nucleated (fish and birds) and non-nucleated (human) red blood cells (RBCs) in order to determine their role in respiration, volume regulation, rigidity/deformability, aging and senescence in physiological and pathological (malaria, cystic fibrosis) conditions.

RBCs are amongst the most frequently used biological models for research on membrane transporters but paradoxically very little was known on ion channels in the RBC membrane due to technical difficulties when we started this activity. We contributed to overcome these difficulties ; our initial studies on fish (Egee et al. 1997; Egee et al. 2000; Egee et al. 1998; Lapaix et al. 2002), bird (Lapaix et al. 2008; Thomas et al. 2001) and human (Bouyer et al. 2006; Bouyer et al. 2007; Decherf et al. 2007; Egee et al. 2002) showed that three types of ion channels are present recursively: K+ channels (activated by Ca2+ ions or ATP-sensitive), non-selective cationic channels and anion channels of variable conductances.

Since 2003 our activity has been almost exclusively (>90%) focused on human RBCs. We figure among the very few groups using electrophysiology for RBCs studies and to our knowledge are the only group for single channel recordings in intact cells placed in ‘physiological’ situations. We have identified the channels activated by Plasmodium falciparum in the host membrane during the intraerythrocytic phase of the malaria parasite cycle (Bouyer et al. 2011a; Bouyer et al. 2006; Bouyer et al. 2007; Egee et al. 2002) and have shown that the parasite up-regulates endogenous channels possibly via phosphorylation by protein kinases (Bouyer et al. 2011b; Merckx et al. 2009; Merckx et al. 2008). We now concentrate our activity on these channels in normal non infected cells.

We have shown recently that membrane deformation (such as induced by contact with a microelectrode) triggers transient activation of the Gardos channel (Ca2+-sensitive K+)(Dyrda et al. 2010) due to sudden increase in Ca2+ permeability. We have demonstrated that the diversity of anion channels activities recorded in previous studies correspond to different levels of activation of a unique type of maxi-anion channel displaying different conductances, kinetics, selectivity and pharmacology depending on conditions(Glogowska et al. 2010). Using Western blotting, mass spectrometry, immunoprecipitation and immunostaining we gave evidence for the presence of a 32 kDa ‘voltage-dependent anion channel’ (VDAC) associated to 18 KDa ‘translocator proteins’ (TSPO) and 30 kDa ‘adenine nucleotide transporters (ANT) in a ‘peripheral benzodiazepin receptor’ (PBR) complex (Bouyer et al. 2011a).VDAC forms the main interface between the mitochondrial and cellular metabolisms in most cell types, and is involved in apoptosis induction.

Most of what we know about its properties was obtained from mitochondrial proteins: voltage-dependent conductance, multiple sub-states with different ion selectivity and permeability. In the open state, VDAC selectively conducts small cations and anions, glutamate, ATP, acetylcholine and dopamine and Tris. The ‘reactive oxygen species’(ROS) and Ca2+ ions were identified as regulators and compounds interacting with Ca2+ binding sites increase the frequency of closure episodes. VDAC is phosphorylated by PKA, PKC and GSK3. What does this mean ? It is stated in handbooks of physiology that the strategy adopted by RBCs for keeping constant the volume of a bag containing ~ 7 mM haemoglobin is to maintain a very low membrane permeability to cations. Therefore, the evidence that the RBC membrane is endowed with a variety of channels capable to allow rapid dissipation of homeostasis by suddenly increasing membrane permeability raises the following question: could there be physiological or physiopathological conditions under which the RBC needs to activate these conductances?

On the basis of our data, our work at present and in the future is aimed at testing a novel concept in which a versatile mechanism involving modulation of the oligomeric structure of VDAC would control channels and pores opening or assembly depending on physiological and pathophysiological conditions. The dynamic equilibrium between VDAC monomeric and oligomeric states could be regulated by factors such as Ca2+, oxidative stress, HK-I, glutamate, NADH, low ATP concentrations, etc… or by interactions with other proteins such as ANT and TSPO or with cytoskeletal tubulin and actin. It is most likely that VDAC interactions with proteins constitutive of or imported in the membrane are responsible for modifications of RBC rigidity/deformability and adhesive properties. It is known that channels are active in specific physiological and pathophysiological situations such as senescence, sickle cell anemia and malaria. These situations have been very well documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. A VDAC channel able to function in different functional states of varying cation or anion selectivity, in parallel with Gardos channel could account for a number of different conditions documented in human RBCs. In addition,even though the state of ‘resting cell’ is the most frequent in ex vivo conditions, it is most likely that the RBC are exposed to more drastic conditions in vivo. It can be approximated that a RBC of healthy adult human must perform a body round trip every single minute. This means that a RBC has to squeeze approximately 160,000 times through pulmonary and tissue capillaries with a diameter lower than its own. There is increasing evidence that, in case of excessive tissue demand, a signaling pathway is activated resulting in the release of ATP acting in a paracrine fashion to increase vascular calibre. The permeability pathways involved in this process are not well defined but it is reasonable to consider that calcium entry through deformation-induced calcium permeability plus Gardos channel activation plus VDAC activation in either of its two modes (cationic or anionic) provide the cell with a fantastic machinery. In addition to matching oxygen delivery with local need, these pathways give the RBC the potential to in situ adapt its own membrane deformability and volume. The coexistence of Gardos channel and VDAC, enabling induction of inverse volume variations, combined with efficient Ca2+-ATPase and Na+/K+-ATPase would then constitute much more an advantage than a threat for RBC homeostasis. At the opposite, a defect in this cascade of events resulting from cell sequestration in vivo or storage in vitro in deleterious conditions could lead to early destruction of RBCs.

Main publications

1. Sanyal S, Egée S, Bouyer G, Perrot S, Safeukui I, Bischoff E, Buffet P, Deitsch KW, Mercereau-Puijalon O, David PH, Templeton TJ, Lavazec C Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties. Blood. 2012 12;119(2):e1-8

2. Bouyer, G., Cueff, A., Egée,S., Kmiecik, J., Maksimova, Y., Glogowska1,E., Gallagher2, P.G., Thomas, S.L.Y. (2011) Erythrocyte peripheral type benzodiazepine receptor /voltage-dependent anion channels are up regulated by Plasmodium falciparum. Blood 118 (8), 2305-2312. (doi:10.1182/blood-2011-01-329300)

3. Thomas, S.L.Y., Bouyer, G., Cueff, A., Egée, S., Glogowska, E., Ollivaux, C. (2011) Ion channels in human red blood cell membrane: actors or relics? Blood Cells, Mol. Dis. 46, 261-265

4. Bouyer, G., Thomas, S.L.Y., Egée, S. (2010) Protein kinase-regulated inwardly rectifying anion and organic osmolyte channels in malaria-infected erythrocytes. Open Biology 4, 10-17

5. Glogowska, E., Dyrda, A., Cueff, A., Bouyer, G., Egée, S., Bennekou, P., Thomas, S.L.Y. (2010). Anion conductance of the human red cell is carried by a maxi-anion channel. Blood Cells, Mol. Dis. 44, 243-251

6. Dyrda, A., Cytlak,U., Ciuraszkiewicz, A., Lipinska, A., Cueff, A., Bouyer, G., Egée, S., Bennekou, P., Lew, V.L., Thomas, S.L.Y (2010). Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells. PLoS One, 5 (2), e9447 (doi:10.1371/journal.pone.0009447)

7. Cueff, A., Seear, R., Dyrda, A., Bouyer, G., Egée, S., Esposito, A., Skepper, J., Tiffert, T., Lew, V.L., Thomas, S.L.Y. (2010). Effects of elevated intracellular calcium on the osmotic fragility of human red blood cells. Cell Calcium 47(1), 29-36 (doi:10.1016/j.ceca.2009.11.002)

8. Merckx, A., Bouyer,G., Thomas,S.L., Langsley,G., Egée, S. (2009). Anion channels in P. falciparum infected erythrocytes and Protein Kinase A. Trends Parasitol. 25(3), 139-144

9. Merckx, A., Nivez, M.P., Bouyer, G., Alano, P., Langsley, G., Deitsch, K., Thomas, S., Doerig C. , Egée, S. (2008). Plasmodium falciparum regulatory subunit of cAMP-dependent PKA and anion channel conductance. PLoS Pathogen, 4(2), e19

10. Bouyer, G., Egee, S. & Thomas, S. L. (2007). Toward a unifying model of malaria-induced channel activity. Proc. Natl Acad. Sci. U S A 104, 11044-9