About the Project
It will rely on a diversity of techniques and approaches (from the diversity of channel expression in progenitors to their involvement in aging and deformability tests) and also include RBC membrane permeability modelling to decipher the involvement of ion channels at these crucial life steps. It will bring implications useful for a better comprehension of pathologies linked to cation permeability, for storage lesions and for the production of cultured RBCs in Bioreactors.
State of the art:
Erythrocytes are highly specialized and atypical cells, as evolution has led to the complete loss of intracellular organelles in mature red cells to optimize respiratory functions. Nonetheless, human erythrocytes are far from being a simple bag of hemoglobin, and have to maintain their deformability, volume and intern homeostasis to accomplish with the best efficiency their function of gas transporters. Indeed, RBCs are in continuous flow, with high and rapid variations of their environment and frequent deformability stresses. This deformability is linked to their unique cytoskeleton and surface / volume ratio, and to a maintenance of the volume; it is tested very regularly in the spleen. During their 120 days of life, RBCs undergo an increase in cell density, reduction of cell volume and are eventually removed from the circulation in the spleen and recycled by macrophages.
Membrane ion permeability is a key parameter for the hydration and volume preservation, and mammalian red cells have long been a paradigm to study membrane transport. Erythrocyte membranes are characterized by an important anionic permeability, allowing the equilibrium for chloride ions and facilitating the transport of CO2 via the Jacobs-Stewart cycle. In contrast, cationic permeability represents a threat linked to the colloido-osmotic pressure exerted by the high protein content of the cell. Surprisingly erythrocytes possess not only anionic channels, but also a repertoire of cationic channels whose full description is still lacking, including notably the Gárdos channel (KCNN4, hSK4) (1), selective for K+ ions, PIEZO-1 (2), a mechanosensitive cation channel or the NMDA Receptor (3). The complete repertoire of ion channel is still under investigation, and so is it for their physiological roles (4, 5). A certitude is that dysregulations of cation channel activity (linked to mutations either on the channels or on partner proteins) are involved in multiple pathologies of the RBC (6, 7) and that these channels are linked to senescence of RBCs (8). The roles of these channels has almost always been studied in static conditions, using electrophysiological techniques.
This project aims to :
1) Fully describe the repertoire of ionic channels from early stages of terminal differentiation to mature RBCs.
2) Understand their physiological role in mature RBCs once they are released within circulation.
3) Unravel their involvement in senescence processes in health and disease conditions.
4) Ultimately all the data will be used to feed a quantitative model (already written and yet improved (9, 10)) for diagnostic predictive purposes, for blood preservation in vitro and for the upscaling of cultured RBC production in bioreactors.
- To determine the repertoire of ion channels in RBCs, we have started a study focusing on the expression of channel proteins in terminal erythropoiesis RBC. All tools are now ready and we have already acquired first results. This part of the project includes erythropoiesis ex vivo, qPCR, Western Blot, and immunofluorescence assays.
- Monitoring of ion channel activity linked to cell flow: We will perform patch clamp studies / CCCP monitoring of membrane potential before and after several stresses implying cell deformation : microsphiltration (11), Shear flow, spleen-on-chip or microfluidic in collaboration with partners from the Evidence ITN.
- model of membrane permeability : the model was originally developed by Lew and Bookchin (9), and was recently updated.
The programme offers highly competitive and attractive salary. The candidate will receive a salary in accordance with the regulations for MSC Fellows/early stage researchers. Exact salary will be confirmed upon appointment but will involve a Living Allowance and monthly mobility allowance depending on the candidate’s personal family situation.
2. Albuisson J, Murthy SE, Bandell M, Coste B, Louis-dit-Picard H, Mathur J, Fénéant-Thibault M, Tertian G, de Jaureguiberry J-P, Syfuss P-Y, Cahalan S, Garçon L, Toutain F, Simon Rohrlich P, Delaunay J, Picard V, Jeunemaitre X, Patapoutian A. Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels [Article]. Nat Commun. 2013;4:1884. doi:10.1038/ncomms2899.
3. Makhro A, Hänggi P, Goede JS, Wang J, Brüggemann A, Gassmann M, Schmugge M, Kaestner L, Speer O, Bogdanova A. N-methyl-d-aspartate receptors in human erythroid precursor cells and in circulating red blood cells contribute to the intracellular calcium regulation. Am J Physiol Cell Physiol. 2013 305(11):C1123-C1138. doi:10.1152/ajpcell.00031.2013.
4. Thomas SL, Bouyer G, Cueff A, Egee S, Glogowska E, Ollivaux C. Ion channels in human red blood cell membrane: actors or relics? Blood Cells Mol Dis. 2011 Apr 15;46(4):261-5. doi:S1079-9796(11)00058-1 PMID 21429775.
5. Hertz L, Huisjes R, Llaudet-Planas E, Petkova-Kirova P, Makhro A, Danielczok JG, Egee S, del Mar Mañú-Pereira M, van Wijk R, Vives Corrons J-L, Bogdanova A, Kaestner L. Is Increased Intracellular Calcium in Red Blood Cells a Common Component in the Molecular Mechanism Causing Anemia? [Perspective]. Frontiers in Physiology. 2017 2017-September-06;8(673). doi:10.3389/fphys.2017.00673.
6. Gallagher PG. Disorders of erythrocyte hydration. Blood. 2017. doi:10.1182/blood-2017-04-590810.
7. Kaestner L, Bogdanova A, Egee S. Calcium Channels and Calcium-Regulated Channels in Human Red Blood Cells. In: Islam MS, editor. Calcium Signaling. Cham: Springer International Publishing; 2020. p. 625-648. doi:10.1007/978-3-030-12457-1_25
8. Lew VL, Tiffert T. On the Mechanism of Human Red Blood Cell Longevity: Roles of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels [Mini Review]. Frontiers in Physiology. 2017 2017-December-12;8(977). doi:10.3389/fphys.2017.00977.
9. Lew VL, Bookchin RM. Volume, pH, and ion-content regulation in human red cells: analysis of transient behavior with an integrated model. J Membr Biol. 1986;92(1):57-74. PMID 3746891.
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