Human RBC membrane in disease 

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Anion conductance of the human red cell is carried by maxi-anion channel (Blood Cell, Molecules and Diseases, 2010).

Edyta Glogowska, Agnieszka Dyrda, Anne Cueff, Guillaume Bouyer, Stéphane Egée, Poul Bennekou, Serge L.Y. Thomas. Contribution: SLYT conceived and designed project and experiments, contributed to their performance, analyzed data and wrote the manuscript; PB analyzed data and revised the manuscript; EG and AD contributed equally to the majority of experi-ments and analysed data; AC, GB and SE contributed to experimental design and to experiments.

Human red blood cells.

Red blood cells (RBCs), also called erythrocytes make up the most plentiful morphotic elements of the blood population, numbering of (4 – 6) x 106 per one mi-croliter of this tissue. From stem cells in bone marrow, human erythroid cells are differentiated through a process named erythropoiesis to become mature erythro-cytes. A typical human erythrocyte measures about 6 – 8 μm in diameter and 2 μm in thickness, and has flattened biconcave shape. Circulating human RBCs are rela-tive simple cells due to the lack of intracellular organelles (mitochondria, ri-bosomes, Golgi apparatus, endoplasmic reticulum, lysosomes) and nucleus. These characteristic properties allow to optimize their two main duties: i) the delivery of oxygen from lungs to tissues, and ii) removal of waste such as carbon dioxide, both caused by increasing in capacity of transported O2 (lack of organelles let more space for haemoglobin, Hb, universal respiratory oxygen-fixing pigment) and area-to-volume ratio (greater for biconcave shape than a sphere of the same diameter). These functions of RBCs are realized by two specialized molecular machines: Hb (normal erythrocytes contain 5 mM of this metalloprotein, constituting 97.5% of the total cell protein by weight) and membrane anion exchange carrier (AE1). From the structural point of view human red blood cells are the simplest of all eu-karyotic cells and this makes them very useful tool for studies on plasma mem-brane transport systems.

Membrane transporters of the human RBCs in health.

Whereas the molecules such as O2 and CO2 pass through the red cell membrane by diffusion according to their partial pressure gradients, organic and inorganic com-pounds (among them ions as the most interesting from the point of this thesis), in-fluencing the electrolyte and acid-base intracellular equilibrium, need other spe-cific pathways. Different transport systems have been characterized in the mem-brane of normal (non-infected) human red blood cells: pumps, using the energy of ATP hydrolysis to transport ions against their electrochemical gradient; channels, specific proteins allowing ions to cross the membrane by the use of passive flow down their electrochemical gradients; cotransporters: antiporters, symporters and uniporters, in which movement of one ion species against its electrochemical gradient is powered by the downhill movement of another (summary in Fig. II. 1).
Interaction of membrane transporters, cytoplasmic buffer (Table II. 1 provides the electrolyte composition of plasma), charge and osmotic properties of haemoglobin and other impermeable solutes assure the control of RBC volume, pH, membrane potential and ion content. Therefore, transporters (together with membrane cy-toskeleton) contribute to maintenance of the cell integrity, its stability and de-formability in response to shear forces of blood circulation.
This work focuses on ionic channels in the human red cell membrane and, because up to-date only cationic channels (Ca2+-activated K+ channel known as a Gardos channel, and non-selective voltage-dependent cationic channel NSVDC, called fur-ther NSC) have been well characterized, it is aimed at describing anionic conduc-tive pathways, clarifying some controversial and unanswered aspects and verifying their physiological role.

Human RBC membrane in disease.

As mentioned, anionic channels in the human red blood cell membrane have been firstly recognized and described, at least partly, in pathological conditions, such as in malaria after Plasmodium falciparum invasion. In contrast to healthy human RBCs, in this situation changes in erythrocyte membrane have been indicated.

Remodelling of the host erythrocyte membrane by Plasmodium falciparum.

Plasmodium falciparum invades mature RBCs nearly metabolically inert and devoid of functional trafficking machinery. From the pathogenetical point of view this may seem like a perfect hideaway from the host’s immune system. However, firstly the erythrocyte’s environment appears for the parasite insufficient. Although the in-tracellular parasite is using most of the nutrients mainly from the digestion of the host haemoglobin (Rosenthal and Meshick, 1998; Krugliak et al., 2002; Lew et al., 2003), several essential components necessary for its growth have to be supplied from the outside of the infected cell. For instance, parasite survival is totally de-pendent on isoleucine (Sherman, 1997) and human haemoglobin does not contain this amino acid. To reach the parasite, this substrate must first cross the host red blood cell plasma membrane (Martin and Kirk, 2007). In some cases, the endoge-nous specific transport systems of erythrocyte membrane (described above) are able to maintain an adequate supply. This takes place, for example, for glucose, the primary energy source for the parasite, transported through endogenous receptor GLUT1. Nevertheless, for some other essential nutrients including, e.g. the vitamin B5 (panthothenic acid) (Saliba et al., 1998) or the amino acid glutamate (Divo et al., 1985) the transport pathways do not exist in the human red cell membrane.

New permeability pathways (NPPs).

Since the 80s, large number of studies tried to describe modifications in membrane permeability after malaria infection. Despite the above-mentioned intracellular conditions, unfavourable for the parasite, it has been demonstrated that the trans-port properties of the P. falciparum infected red cell membrane become obviously different from the non-infected cells (Staines and Kirk, 1998; Staines et al., 2002): i) 100-fold increase of the glycolitic rate of the infected RBCs, ii) generation of new metabolic processes, iii) increase in the traffic of nutrients, waste products and cations, iv) dissipation of the normal Na+ and K+ gradients across the host cell membrane. The question was which strategies is malaria parasite using to be able to grow and replicate without the red blood cell swelling and bursting prema-turely? It has been shown, that when red cell endogenous system do not exist or cannot maintain proper supply, the parasite activates other transporters, referred as ‘New Permeability Pathways’ (NPPs), approximately 10 – 20 h post-invasion (hpi), thus allowing permeation of low-molecular-weight solutes. Radiotracer flux and haemolysis experiments performed by Hagai Ginsburg and Kiaran Kirk indi-cate functional and pharmacological properties of NPPs (Ginsburg et al., 1983; Ginsburg et al., 1985; Ginsburg, 1994; Ginsburg and Kirk, 1998; Kirk et al., 1994; Kirk et al., 1999; Kirk, 2001). They characterized the nature of the transported substrates, their rates of transport, selectivity properties and inhibitors of these new pathways, as follow: NPPs allow both organic and inorganic anions (i.e., negatively charged lac-tate or Cl- ions), electroneutral molecules (i.e., polyols, amino acids, nucleo-sides), and organic and inorganic cations (i.e., positively charged Na+ and K+) to pass; they display preferentially anionic selectivity; indicated permeability for solutes is presented in Fig. II. 6; NPPs are sensitive for the known anion-selective transport pathways block-ers such a: 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), fu-rosemide, niflumic acid and glybenclamide.

Electrophysiological studies on anionic channels in human RBCs.

For a long time, the question of whether NPPs represent endogenous channels up-regulated and/or modified by the parasite or either they are parasite engendered has been around and still remains unanswered. The origin of the NPPs was quite controversial and they have been suggested to be both parasite and host cell de-rived (reviewed by Kirk, 2001). On the other hand, the techniques available to study these conductive pathways were poor. For instance, taking into account how little protein represent a few hundred channels the standard proteomic protocols for detection of these channels were very limiting. However, since parasite-induced transport pathways for anions and other solutes in infected RBCs showed importance in transport studies and since NPPs demonstrated many similarities to anion-selective channels, the patch-clamp electrophysiological technique repre-sented the best method available to investigate channel-mediated transport of charged solutes through the infected red blood cell membrane (description of this technique: section III. 7, Materials and Methods).
Using the patch-clamp electrophysiological technique, S. Desai and co-workers in Bethesda demonstrated that the membrane conductance of infected RBCs is 150 times greater than that measured in non-parasitized RBCs and that this increased conductance results from activation of small anionic channels showing functional and pharmacological properties of NPPs (Desai et al., 2000). In addition, they re-ported that these channels had better open probability at negative potentials which correspond to inwardly rectified conductance. Moreover, identified chan-nels have shown anion selectivity I- > Br- > Cl- > lactate-. Furthermore, they named these channels PSAC (Plasmodial Surface Anionic Channels) and suggested that NPPs have a parasite origin and are channels formed from parasite derived pro-teins (Alkhalil et al., 2004). In recent study (Nguitragool et al., 2011) they have identified protein which is exported after P. falciparum infection to the host eryth-rocyte and plays a role in PSAC activation. They gave evidence for this parasite-encoded CLAG3 protein as a key component contributing to solute uptake in in-fected RBCs.

Table of contents :

LIST OF ABBREVIATIONS
LIST OF FIGURES
LIST OF TABLES
I. IN BRIEF 
I. 1. Aim of the study
I. 2. Global context and objectives
I. 3. Summary of results
I. 4. Scientific communication
I. 4. 1. Publications
I. 4. 2. Presentations on international conferences
I. 5. IN BRIEF en Français
II. THEORETICAL BACKGROUND 
II. 1. Human red blood cells
II. 2. Membrane transporters of the human RBCs in health
II. 2. 1. Exchangers, pumps, cotransporters
II. 2. 2. Ionic channels
II. 2. 2. 1. Cationic channels
II. 2. 2. 2. Anionic channels
II. 3. Human RBC membrane in disease
II. 3. 1. Infection of the human RBCs by malaria parasite
II. 3. 2. Remodelling of the host erythrocyte membrane  Plasmodium falciparum
II. 3. 2. 1. New permeability pathways (NPPs)
II. 3. 2. 2. Morphological changes
II. 4. Electrophysiological studies on anionic channels in human RBCs
II. 5. Molecular nature of anionic channels
II. 6. Physiological role of human red cell membrane channels in health and disease
III. MATERIALS AND METHODS 
III. 1. Red blood cells
III. 2. Malaria infected red blood cells
III. 3. Magnetic separation of malaria infected red blood cells
III. 3. 1. Principle
III. 3. 2. Protocol
III. 4. Haemolysis of RESA1 P.falciparum-infected human erythrocytes in isosmotic sorbitol solution
III. 4. 1. Principle
III. 4. 2. Protocol
III. 5. Western blotting of RESA1 P.falciparum-infected human erythrocytes
III. 5. 1. Cells preparation
III. 5. 2. Samples preparation
III. 5. 3. Extraction and denaturation
III. 5. 4. Electrophoresis and blot
III. 6. Immunofluorescence staining and confocal microscopy of RESA1 P.falciparum-infected human erythrocytes
III. 7. Patch-clamp
III. 7. 1. Principle
III. 7. 2. Current recordings
III. 7. 3. Current analysis
III. 8. Percoll-gradient separation of human red blood cells
III. 8. 1. Principle
III. 8. 2. Protocol
IV. RESULTS 
IV. 1. First objective: Further clues on electrophysiological characterization of anionic channels in human red cell membrane
IV. 1. 1. Introduction
IV. 1. 2. Results
IV. 1. 3. Discussion
IV. 1. 4. Article
IV. 2. Second objective: The molecular identity and regulation of anionic channels in the physiology and pathophysiology of the human red blood cells
IV. 2. 1. Introduction
IV. 2. 2. Results
IV. 2. 3. Discussion
IV. 2. 4. Article
IV. 3. Third objective: The activation of anionic channels by Plasmodium falciparum and possible involvement of RESA1 protein in this process
IV. 3. 1. Introduction
IV. 3. 1. 1. Ring infected Erythrocyte Surface Antigen (RESA)
IV. 3. 1. 1. 1. Structure
IV. 3. 1. 1. 2. Link with spectrin
IV. 3. 1. 1. 3. Role in malaria infected erythrocyte
IV. 3. 2. Results
IV. 3. 3. Discussion
IV. 4. Forth objective: Physiological role of erythrocyte channels: A unifying hypothesis of senescence, sickle cells and malaria
IV. 4. 1. Introduction
IV. 4. 2. Results
IV. 4. 3. Discussion
V. GENERAL CONCLUSIONS AND PROSPECTS 
V. 1. Concluding remarks
V. 2. Discussion and perspectives
REFERENCES 
ANNEX
ACKNOWLEDGMENTS

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