Contribution of ionic channels in dehydration-elicited rehydration

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Unlike plants, animals are unable to use inorganic matter to build organic compounds. Instead, most of them find energy through the oxidation of organic substrates which they obtain by eating plants or other animals. When Lavoisier and Laplace claimed in 1780 that “Life is combustion”, they simply characterized respiration as consumption of oxygen (O2) and production of carbon dioxide (CO2) with liberation of heat. However, in contrast to combustion of a piece of charcoal which produces carbon dioxide by oxygen binding to carbon, in the process of animal respiration oxygen does not bind to carbon to produce carbon dioxide. Rather, in intracellular energy-yielding reactions, the oxidation of organic compounds liberates electrons and oxygen supplied by respiration is used as an acceptor of this electrons, oxygen fixes them in parallel with formation of the usual end-products of oxidation of organic substrates in animals: carbon dioxide and water.


In unicellular organisms and many small metazoa, oxygen is transferred exclusively in solution, but in most complex metazoa oxygen fixing pigments are necessary vehicles for transferring these molecules by diffusion or by convection through various structures and compartments between the ambient media, air or water, to the cells. Oxygen-fixing pigments have a very long evolutionary history (for a review, see (Scott 1966; Glomski and Tamburlin 1989; Glomski and Tamburlin 1990). Among them, haemoglobin (Hb) molecules appeared more than 1000 million years ago and have become established as the dominant universal respiratory pigment of vertebrates. In this metalloprotein, the haeme moiety of the molecule can be considered evolutionary stable and is responsible for the reversible binding of oxygen, while the globin moiety is structurally variable and, through this variability, determines the conditions under which oxygen binding and release can be favoured. Hb molecules are therefore specifically adapted for species and for environmental or temporal requisites.


In vertebrates, Hb molecules are enclosed in circulating erythrocytes which offer the important advantage to separate the chemical intraerythrocytic environment from the plasma and then a better control of factors affecting oxygen binding and release such as organic phosphates and inorganic ions. RBCs have then evolved to optimize two functions: (1) the transport of oxygen from lungs or gills to tissues, and (2) removal of waste such as carbon dioxide. This job is done by two specialized molecular machines: Hb and an anion exchange carrier (AE1) in the RBC membrane. RBCs from most species are richly endowed with these two components (Annex #1). All the other components are geared to maintain the constancy of volume and elastic properties that allow RBCs to bend and flow through the narrowest of capillaries (Annex #2 and #3).

human red blood cell

Human erythrocytes are the most abundant morphotic element of blood. One microliter of this tissue contains (4 – 6) x 106 RBCs. Human RBCs are small (~8 µm in diameter and ~2 µm in thickness which gives ~90 fl of cell volume) and biconcave in form. This characteristic shape is a consequence of cell maturation. In this process, the nucleus is extruded, mitochondria, endoplasmic reticulum, ribosomes, Golgi apparatus and lysosomes are degraded. Thus circulating human RBCs are devoid of intracellular organelles. This increases both capacity of transported O2, because there is more space for Hb molecules and the area-to-volume ratio (A/V), which is ~40% higher if compared to a sphere of the same diameter. There are ~270 millions of Hb molecules in one RBC,  which is equal to 340g/loc or 5 mM of this metalloprotein.

RBC membrane transporters

As a consequence of haemoglobin encapsulation, the transport of O2 and CO2 within the blood is intricately related to the electrolytes and acid-base status of the RBCs, which are themselves strongly dependent on the permeability properties of the membrane. Indeed, whereas O2 and CO2 are able to cross the cell membranes by solubility/diffusion processes according to their gradients of partial pressure, the organic and inorganic substances influencing the electrolytes and acid-base status diffuse very slowly across the lipid bilayer of the membrane and therefore must follow specific pathways. There are eventually a limited number of means by which solutes move across the RBC membrane and thus influence the gaseous exchanges. This work focuses on electrodiffusional ionic movements through RBC membrane exclusively and is aimed to underline the main characteristics of conductive pathways in human RBC and to address some of the more controversial and poorly understood aspects of their regulation and physiological role. These aspects are considered in the double standpoint i/ of the implication of ionic movements in upholding the electrolytic and acid-base steady-state corresponding to intracellular homeostasis, and ii/ of their participation in the processes of regulation activated as soon as this steady-state is disrupted.

pumps, exchangers, cotransporters

Among the different types of ion transports known in animal cells, those documented up to now in RBCs result from the activity of transporters belonging to two major families: 1/ the primary active and energy consuming Na+/K+ ATPase and Ca2+ ATPase and 2/ the secondary active transporters, labelled as exchangers (or antiports) and cotransports (or symports) according to the relative direction of the solutes. The Na+/K+ ATPase is an electrogenic pump whilst the transport processes of the second family (Na+/H+ exchanger, Cl-/HCO3- exchanger and K+-Cl- cotransporter) are electrically silent. Most of the above ion pathways have been observed in all vertebrates with few exceptions and are shared by both anucleated and nucleated RBCs. However, many other ion pathways exist and have been described in many vertebrates, particularly ionic channels are present in human, avian, batracian and fish RBCs (Thomas and Egee 1998; Shindo et al. 2000; Lapaix et al. 2002; Hamill 2006; Lapaix et al. 2008).


In RBCs water exchange is ~4 ms what is equal to diffusional water permeability (Pd) 4 x 10-3 cm/s. But the ratio of osmotic (Pf) to diffusional water permeability is Pf:Pd = 6.
“The discovery of aquaporin water channels (AQP) in the early 1990s (Denker et al. 1988; Smith and Agre 1991) has, to a great extent, solved the paradoxes surrounding water permeability in a variety of eukaryotic cell types. AQP1, the first water channel to be identified at the molecular level from RBCs, is 28 kDa protein. It was shown that it is an integral membrane protein and present in erythrocytes at a high copy number (typically (12000 – 160000) copies/cell). Furthermore two forms of the protein, 28 kDa and (35 – 60) kDa (HMW-28 kDa) were detected and this second one was shown to be an N-glycosylated form of 28 kDa.
Erythrocytes express at least two isoforms of AQP, namely 1 and 3. Of these, AQP1 is by far most abundant. AQP3 is resistant to the action of mercurial agents and this, therefore, explains the small mercury-insensitive component of erythrocyte water permeability. Although AQP3 demonstrates a high permeability to glycerol and urea, it must, however, be remembered that specific, facilitative urea transporters exist in erythrocyte membranes which also contribute to the high urea permeability of this cell type.
Opinions vary as to why RBCs require such a high permeability to water. First – cirucalting RBCs encounter region of higher than 1 M osmolality (the renal medulla area). So the rapid unload and load water is desired. Second – recent studies demonstrated that AQP1, in common with certain other water channels, is additionally permeable to CO2. But individuals lacking AQP1 expression (Colton group-null individuals) demonstrate no clinical abnormalities and no significant erythrocytes abnormality, apart from a significantly reduced osmotic water permeability (~80%) nor are significant changes in CO2 gas transport noticeable. So the physiological role of AQP is not clear.” (Browning and Wilkins 2003).

ionic channels

The information available when this work was undertaken on ion movements in human RBCs were provided mostly by tracer flux studies and were therefore limited for giving molecular details on the nature of the ion pathways. The technique of extracellular patch clamp has little been used in human RBCs, even though the pioneer works of (Hamill 1981) have shown that its high temporal and spatial resolution provide a unique opportunity to extend the knowledge of RBC permeabilities to the molecular level. The patch clamp electrophysiological technique represents the best method available to investigate channel-mediated transport of charged solutes through the red cell membrane. By measuring the electrical current across a membrane (produced by the charged solutes) in relation to a known applied potential difference, it is possible to obtain a detailed characterisation of ion transporting channels at both cellular and single level. However, until recently, electrophysiological studies on human RBCs have been difficult due to the fragility and small size of the cells, and little is known of the anionic conductive pathways present in the RBC membrane in health and disease. The work on human red cell is in marked contrast to early studies on nucleated, e.g. amphibian (Hamill 1983) and more recently fish (Egee et al. 1998; Lapaix et al. 2002) and avian (Drew et al. 2002; Lapaix et al. 2008) red cells where patch clamping proved easy and productive. The difference was attributed to the size of these cells.

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cationic channels

During the past two decades, electrophysiological studies on human RBCs revealed two different cationic channels: an intermediate conductance Ca2+-activated K+ channel, known as the Gardos channel from the name of this Hungarian scientist who first showed that metabolic depletion/arrest induces K+ efflux when Ca2+ ions are present in the extra-cellular medium (Gardos 1958), and a non-selective cationic (NSC) channel. Many attempts have been made to characterise Ca2+-gated K+ channels in human erythrocytes. It has been shown that metabolic depletion (Gardos 1958) is not the only activator of Gardos channels. Many others activators as well as modulators have been reported to impact this conductive pathway (Annex #4). Having various modes of action, these compounds alter the hIK1 channels directly or indirectly. The action of three compounds should be underlined: clotrimazole (CLT), charibdotoxin (ChTX) and apamine. The two first, CLT and ChTX, are potent Gardos channel inhibitors whereas apamine is not. Because of this pharmacological characteristic, intermediate Ca2+- dependent K+ channels can be distinguished form small Ca2+-gated K+ channels. Based on patch clamp measurements, mainly in excised I-O configuration, Gardos channel has been classified as a highly-K+ selective, inwardly rectified, voltage-independent (Tharp and Bowles 2009), with a conductance of ~20 pS. To activate this channel, requirement of one (Leinders et al. 1992) or two (Grygorczyk and Schwarz 1983; Schwarz and Passow 1983; Grygorczyk and Schwarz 1985) Ca2+ ions have been reported. But it should be kept in mind that Ca2+ ions do not interact with the channels’ protein directly but through camodulin (CaM). CaM is a small (~16 kDa), acidic protein possessing high affinity motifs for Ca2+ binding. In Gardos channels CaM is prebound to the cytoplasmic C-tail of the channel and mediates Ca2+-dependent gating of these channels. As Fanger and co-workers demonstrated, four calmodulin molecules are associated with the channel and Ca2+ ions must be bound to all four CaM proteins to induced conformational changes for the channel to open (Fanger et al. 1999). Thus, not 1 or 2 but 4 Ca2+ ions are required to activate KCa 3.1. As shown by Stampe and Vestergaard-Bogind (Stampe and Vestergaard-Bogind 1985), the KCa 3.1 sensitivity to intracellular ionized Ca2+ changes with the intracellular pH (pHi) variation. In their experimental conditions half-maximal K+ efflux was observed at pHi 6.1. However, this pHi value was shifted to higher pHi at lower ionized Ca2+ concentration (Stampe and Vestergaard-Bogind 1985).
Furthermore Gardos channel’s open probability (Po) seems to be dependent on temperature (increases with temperature reduction) (Grygorczyk 1987) and [Ca2+]i (increasing with [Ca2+]i increase) (Leinders et al. 1992) but Po is membrane potential-independent (Leinders et al. 1992). For interpretation of recordings presented in the results section, it is important to keep in mind that in most cases extinction of Gardos channel activity is manifested by Po decline rather than by current amplitude reduction (exception –inhibition by TEA) (Dunn 1998). According to different authors the number of channels per red cell have been estimated to be ~10 copies/RBC (Grygorczyk and Schwarz 1983), 55 copies/RBC (Grygorczyk et al. 1984) or, more recently, (100 – 200) copies/RBC (Lew et al. 1982; Alvarez and Garcia-Sancho 1987).
Although increased intracellular Ca2+ is necessary in Gardos channel mediated K+ efflux, Romero and co-workers have suggested that the channel behaviour remains under complex metabolic control (Romero and Rojas 1992). This additional control presumably would involve cAMP-mediated and non-mediated mechanisms, and among them direct ATP–channel interactions and/or phosphorylation of the channel proteins. Also recent studies of Tharp and colleges, which are based on analysis of the KCa 3.1 sequence, pointed on phosphorylation of C-terminus of the channel’s protein as an activator of K+ movement across the membrane (Tharp and Bowles 2009). However, the results of Pellegrino and Pellegrini studies (Pellegrino and Pellegrini 1998) are in opposition to above mentioned statement. Using patch clamp technique in I-O configuration it has been demonstrated that in contrast to Romero’s work, Gardos channel is up-regulated by endogenous PKA. The enhancement of channel activity by cAMP stimulation was also found. In spite of these discrepancies, both groups hypothesised the presence of two types of Ca2+-dependent K+ channels in human erythrocytes. According to Low and co-workers, peroxiredoxin 2 (Prx2), a part of intracellular erythrocyte’s antioxidant system, modulates the Gardos channel activity (Low et al. 2008).

Table of contents :

I. 1. Aims.
I. 2. Summary of results.
I. 3. Scientific communication.
I. 3. 1. Publications.
I. 3. 2. Oral communications.
I. 4. IN BRIEF en Fraçais.
I. 5. IN BRIEF po polsku.
II. 1. Oxygen.
II. 2. Haemoglobin.
II. 3. Erythrocytes.
II. 4. Human red blood cell.
II. 5. RBC membrane transporters.
II. 5. 1. Pumps, exchangers, cotransporters.
II. 5 .2. Aquaporins.
II. 5. 3. Ionic channels.
II. 5. 3. 1. Cationic channels.
II. 5. 3. 2. Anionic channels.
II. 5. 3. 3. Physiological role of ionic channels.
III. 1. Contribution of ionic channels in dehydration-elicited rehydration: lysis curves and osmotic fragility data acquisition.
III. 1. 1. Experimental design.
III. 1. 2. Solutions.
III. 1. 3. Preparation of red blood cells.
III. 1. 4. The biphasic OFC protocol.
III. 1. 5. Measurement of the volume-fraction of RBC cytosol lost by Ca2+-induced exovesiculation*.
III. 1. 5. 1. Processing of vesicle samples for transmission and scanning electronmicroscopy.
III. 2. Electrophysiology.
III. 2. 1. Preparation of cells.
III. 2. 2. Solutions.
III. 2. 3. Current recordings.
III. 3. Chemicals.
III. 4. Red blood cell model.
IV. 1. First question: Are RBC’s membrane channels involved in rehydration elicited by isosmotic dehydration?
IV. 1. 1. Results.
IV. 1. 2. Discussion.
IV. 2. Second question: Does membrane deformation induce channel activity?
IV. 2. 1. Results.
IV. 2. 1. 1. Evidence for spontaneous channel activity following seal formation.
IV. 2. 1. 2. Transient nature of recorded currents.
IV. 2. 1. 3. Identification of Gardos channels.
IV. 2. 1. 4. Simulation by mathematical RBC model.
IV. 2. 1. 5. Evidence for anionic channel activation.
IV. 2. 2. Discussion.
IV. 2. 2. 1. Activation of Gardos channels.
IV. 2. 2. 2. Activation of anionic channels.
IV. 2. 2. 3. Transient nature of Gardos channel activity.
IV. 2. 2. 4. Fast transitions
IV. 3. Third question: What is the molecular identity of anionic channels present in RBC membrane?V. GENERAL CONCLUTIONS AND PROSPECTS.



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