Membrane transporters of the human RBCs in health

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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.
Figure II. 1: Overview of the principal transport pathways of the human red blood cell mem-brane. Ions transport across the red cell membrane is realized by specific mechanisms: pumps (us-ing 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 elec-trochemical gradients) and cotransporters: antiporters, symporters and uniporters (movement of one ion species against its electrochemical gradient is powered by the downhill movement of an-other). The principal transporters in the human RBCs are shown. Cationic (Ca2+-activated K+ chan-nel known as a Gardos channel, and non-selective cationic channel NSC) and anionic channels are pointed out. Up to-date only cationic channels have been well characterized.

THEORETICAL BACKGROUND.

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.

Exchangers, pumps, cotransporters.

Among the different types of RBC membrane transporters, the major protein Cl-/HCO3- anion exchange carrier AE1 (also called band 3, SLC4A1) plays an essential role in the increasing of blood CO2-carrying capacity and supports acid-base ho-meostasis (LaCelle and Rothsteto, 1966; Gunn et al., 1973; Cabantchik, 1999). Due to the strong interaction with lipids and proteins of membrane cytoskeleton it as-sures mechanical integrity and viscoelasticity of RBCs, allowing them resistance to the shear forces of circulation and squeeze through the narrow capillaries (Jay, 1996).
Maintenance of the cell volume is mainly realized by well characterized primary active and energy consuming pumps: Na+/K+ ATPase and Ca2+ ATPase. Indeed, pump-leak mechanism (Tosteson and Hoffman, 1960) sustains a high intracellular K+ concentration (around 140 mM) and low intracellular Na+ concentration (around 5 mM), by pumping sodium out of the cell and potassium into the cell and thus generating electrochemical gradients for both ions (see Table II. 1 for electro-lytes value). In steady-state cytoplasmic Na+/K+ ratio is 0.12 – 0.16 (Bernstein, 1954). The ‘leak’ of sodium and potassium results from other exchangers, trans-porters and channels present in the red cell membrane.
The electrolytes gradient can be used by erythrocytes to facilitate the movement of different solutes through the membrane via secondary active transporters, labelled as cotransporters (symporters or antiporters according to the relative direction of solutes). The Na+/H+ exchanger is an example of such transporter, which plays a key role in the regulation of intracellular pH, using the energy of the Na+ gradient to extrude H+ (Kaloyianni et al., 2001).
Furthermore, other shown principal membrane transporters are: aquaporin 1 (a water channel), K+-Cl- cotransporter, a Na+-2Cl–K+ cotransporter, some amino ac-ids transporters, a glucose transporter (Glut1), an oxidized gluthatione (GSSG) transporter (GSTP), a nucleoside transporter (NT1), choline, lactate, urea … trans-porters.
The human erythrocyte possesses also conductive pathways for ions which are cationic and anionic channels.

Ionic channels.

For a long time the studies on ions passing the red cell membrane through the channels were very poor, due to the limitations of available techniques. Most of the early information about these conductive pathways came from flux experiments, unfortunately restricted in molecular details. The knowledge of RBC membrane permeabilities at the molecular level has evolved due to the successful application of the patch-clamp electrophysiological technique, allowing research on channel-mediated transport of charged solutes across erythrocyte membrane. This led to characterization of ionic channels in nucleated erythrocytes e.g. fish (Egee et al., 1998; Lapaix et al., 2002), and avian (Lapaix et al., 2008). In contrast, human red blood cells brought more difficulties caused by the fragility and a small size of these cells limiting the use of whole-cell recordings.

Cationic channels.

However, in the human red cell membrane two different cationic channels have been characterized:
Ca2+-sensitive K+ channel (also called Gardos channel, Hamil, 1981; Gry-gorczyk et al., 1984; Grygorczyk and Schwarz, 1985; Shields et al., 1985; Stampe and Vestergaard-Bogind, 1985; Alvarez and Garcia-Sancho, 1987; Grygorczyk, 1987; Fehlau et al., 1989; Bennekou and Christophersen, 1990; Christophersen, 1991; Leinders et al., 1992; Romero and Rojas, 1992; Pellegrino and Pellegrini, 1998; Pellegrino et al., 1998; Fanger et al., 1999; Del Carlo et al., 2003; Low et al., 2008; Tharp and Bowles, 2009); non-selective cationic channel (NSC) (Christophersen and Bennekou, 1991; Bennekou, 1993; Kaestner et al., 1999; Kaestner et al., 2000; Huber et al., 2001; Kaestner and Bernhardt, 2002; Duranton et al., 2002; Lang et al., 2003; Rodighiero et al., 2004).
By using the patch-clamp technique the activity of another cationic conductance has been also depicted, corresponding to a non-selective voltage-dependent cati-onic channel, proposed as a nicotinic type of acetylcholine receptor (Bennekou, 1993) and activated at depolarized membrane potential. On the other hand, it has been shown that oxidation of the RBC membrane or energy depletion (Duranton et al., 2002) stimulates this channel. Further, NSC can be activated by removal of in-tracellular and extracellular Cl- (Huber et al., 2001; Duranton et al., 2002; Rodighiero et al., 2004), by incubation in low ionic strength (LIS) medium (LaCelle and Rothsteto, 1966; Jones and Knauf, 1985; Bernhardt et al., 1991) or by pros-taglandine PGE2 (Kaestner et al., 1999; Kaestner and Bernhardt, 2002; Kaestner et al., 2004). The non-selective cationic channel has been reported to be permeable to mono- and divalent cations, especially Ca2+ (Kaestner et al., 2000; Huber et al., 2001; Duranton et al., 2002) and inhibited by amiloride, EIPA and gadolinium (Huber et al., 2001; Duranton et al., 2002; Lang et al., 2003). However, the exact molecular nature of such cationic channel is not completely solved, especially NSVDC and NSC moiety.

Anionic channels.

Already 50 years ago, Dan Tosteson calculated the anion self-exchange rate and pointed alternative pathways for Cl- passing the red cell membrane different from its free electrodiffusion (Tosteson and Hoffman, 1960). However, as a result of technical difficulties related to the small size of erythrocytes and their remarkable deformability these conductive pathways in the human red cell membrane were almost out of reach. Whereas tracer flux experiments showed membrane perme-ability of ~ 10-7 cm/s corresponding to anionic channels numbering in 10-4 –10-6 of the total chloride exchange, just a few electrophysiological reports sug-gested their presence in the human red cell membrane.
These experiments brought however important informations to this subject. For instance, the membrane potential has been defined for about -10 to -12 mV, close to the Nernst potential for chloride (Hoffman and Laris, 1974). This was in agree-ment with a theory that diffusible anions are distributed in accordance with a Donnan equilibrium and that the RBC membrane was relatively impermeable to cations compared to anions (Warburg et al., 1922; Van Slyke et al., 1923; Funder et al., 1966). Limitation by anions of the salt efflux from the RBC has been shown by experiments using components (gramicidin, valinomycin) increasing the mem-brane permeability for cations (Harris and Pressman, 1967; Scarpa et al., 1970). They provided a model of the two-parts anion transporter: i) a large electroneutral exchanger fundamental to the CO2-carrying capacity of the blood (Dalmark and Wieth, 1972; Gunn et al., 1973), and ii) a smaller electrogenic component that de-termines the RBC resting potential (Hunter 1971; Lassen et al., 1978) and could be important as the rate-limiting step for electrolyte and water movements through the RBC membrane.
The exchange concept has been strongly confirmed by Hunter, who showed the net permeability (conductance) 4 orders of magnitude smaller than the tracer perme-ability (Hunter, 1971; Hunter, 1977). The same author estimated the human RBC anionic permeability proper (conductance) of about 10 μS/cm2. A similar value was obtained by Hoffman and co-workers (Hoffman et al., 1980) measuring mem-brane potential- dependent fluorescence, and flux from the electrogenic fraction of the Na+/K+-pump.
Furthermore, in 1972 Cabantchik and Rothstein identified a 100 kDa red cell membrane protein, called band 3 (AE1, SLC4A1) as the pathway for anion trans-port (Cabantchik and Rothstein, 1972). In spite of differences in pH dependence, energy of activation, selectivity, etc… both components, i.e. the electroneutral an-ion exchange and the conductance proper have been attributed to be mediated by band 3. Concerning the mechanism of the assumed conductive pathway it has not almost out of reach. Whereas tracer flux experiments showed membrane perme-ability of ~ 10-7 cm/s corresponding to anionic channels numbering in 10-4 –10-6 of the total chloride exchange, just a few electrophysiological reports sug-gested their presence in the human red cell membrane.
These experiments brought however important informations to this subject. For instance, the membrane potential has been defined for about -10 to -12 mV, close to the Nernst potential for chloride (Hoffman and Laris, 1974). This was in agree-ment with a theory that diffusible anions are distributed in accordance with a Donnan equilibrium and that the RBC membrane was relatively impermeable to cations compared to anions (Warburg et al., 1922; Van Slyke et al., 1923; Funder et al., 1966). Limitation by anions of the salt efflux from the RBC has been shown by experiments using components (gramicidin, valinomycin) increasing the mem-brane permeability for cations (Harris and Pressman, 1967; Scarpa et al., 1970). They provided a model of the two-parts anion transporter: i) a large electroneutral exchanger fundamental to the CO2-carrying capacity of the blood (Dalmark and Wieth, 1972; Gunn et al., 1973), and ii) a smaller electrogenic component that de-termines the RBC resting potential (Hunter 1971; Lassen et al., 1978) and could be important as the rate-limiting step for electrolyte and water movements through the RBC membrane.
The exchange concept has been strongly confirmed by Hunter, who showed the net permeability (conductance) 4 orders of magnitude smaller than the tracer perme-ability (Hunter, 1971; Hunter, 1977). The same author estimated the human RBC anionic permeability proper (conductance) of about 10 μS/cm2. A similar value was obtained by Hoffman and co-workers (Hoffman et al., 1980) measuring mem-brane potential- dependent fluorescence, and flux from the electrogenic fraction of the Na+/K+-pump.
Furthermore, in 1972 Cabantchik and Rothstein identified a 100 kDa red cell membrane protein, called band 3 (AE1, SLC4A1) as the pathway for anion trans-port (Cabantchik and Rothstein, 1972). In spite of differences in pH dependence, energy of activation, selectivity, etc… both components, i.e. the electroneutral an-ion exchange and the conductance proper have been attributed to be mediated by band 3. Concerning the mechanism of the assumed conductive pathway it has not

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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.

Infection of the human RBCs by malaria parasite.

From the four known intracellular protozoan parasites of the genus Plasmodium: P. falciparum, P. vivax, P. ovale and P. malariae which caused an endemic disease called malaria transmitted by the female Anopheles mosquito, the first one, invad-ing the host human RBCs during its asexual life cycle, is responsible for enormous morbidity and mortality. Infection of the human erythrocytes by malaria parasite is a complex and dynamic process. The invading forms of P. falciparum called merozoites interact with the red cell membrane by their ‘apical’ end consisting in specialized secretory organelles, such as the micronemes, rhoptries and dense granules. This leads to erythrocyte membrane deformation and formation of a sta-ble parasite-host cell junction (Dvorak et al., 1975; Marti et al., 2005; see Fig. II. 2). Microneme proteins are mainly involved in initiating invasion, rhoptry and dense granule proteins drive vacuole formation and are implicated in establishment of the parasite in the newly invaded host cell (Cowman and Crabb, 2006). Invagina-tion of the erythrocyte bilayer results after in establishment of the intracellular parasite surrounded by a vacuolar membrane (PVM, parasitophorous vacuole membrane), in its ‘ring’ stage in the host (Bannister et al., 2000). Once the mero-zoite has entered the erythrocyte, it differentiates from ring form to trophozoite (~ 15 h after invasion). At this stage, the erythrocyte is loosing its smooth biconcave discoid shape to become more spherical and small electro-dense protrusions on its surface called ‘knobs’ are formed (Sherman et al., 2004). The parasite then enters the schizont stage corresponding to a rapid DNA/RNA amplification phase leading to the formation of 8 to 32 daughter merozoites. The infected erythrocytes finally rupture and release merozoites ready to invade new red blood cells. Asexual life cycle of P. falciparum (Fig. II. 3) takes approximately 48 h.
It should be also noticed, that inside the red blood cells, the malaria parasite de-velop either into an asexual forms (ring, trophozoite, schizont) or a sexual (micro-and macrogametocytes, males and females, respectively). This strategy helps the parasite to protect from the harmful or lethal effects of antibodies or immune de-fence mechanisms of host.
During invasion and intracellular development of malaria parasite different mor-phological changes accompanied by metabolic and biosynthetic activity alterations occur in the host erythrocyte.

Table of contents :

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 by 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
ACKNOWLEDGMENTS