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Patch-Clamp Analysis of Membrane Transport in Erythrocytes

TL;DR: This chapter describes the main properties of the erythrocyte’s membrane transport system, how electrophysiological techniques can be applied, and how they have contributed to the comprehension of ery throatcyte physiology with the description of the various ion channels that can be found in RBC membrane.
Abstract: Among all the models used to study membrane transport, erythrocytes (Red Blood Cells, RBCs) have probably been the most utilised cell type. Radioisotopes fluxes, isosmotic haemolysis, ion content analysis (e.g. flame photometry), or fluorescence techniques have been widely used to characterise the various transporters present in the RBCs membrane. These techniques have allowed the description of several types of transporters such as pumps, specific solute transporters, symporters or antiporters, and even ion channels. However, the physiology of RBCs and their maintenance of homeostasis remains incompletely understood, and electrophysiology has proven, since the first single-channel recording on a human erythrocyte membrane thirty years ago, to be a very useful tool to understand more deeply RBC membrane transport. Why does one use these techniques on a small, non-excitable cell that has long been considered no more than an empty bag of haemoglobin? The diversity of transporters in the RBC membrane, including ion channels, shows that these cells are much more complex than expected. Indeed, ion channels now described in the RBC membrane (from Mammals to other Vertebrates) are implicated in important phenomena and functions throughout the cells lifespan (gas transports, cell volume regulation, differentiation and death). In this chapter, we will describe the main properties of the erythrocyte’s membrane transport system, how electrophysiological techniques can be applied, and how they have contributed to the comprehension of erythrocyte physiology with the description of the various ion channels that can be found in RBC membrane.

Summary (6 min read)

1. Introduction

  • Among all the models used to study membrane transport, erythrocytes (Red Blood Cells, RBCs) have probably been the most utilised cell type.
  • These techniques have allowed the description of several types of transporters such as pumps, specific solute transporters, symporters or antiporters, and even ion channels.
  • The physiology of RBCs and their maintenance of homeostasis remains incompletely understood, and electrophysiology has proven, since the first single-channel recording on a human erythrocyte membrane thirty years ago, to be a very useful tool to understand more deeply RBC membrane transport.
  • The diversity of transporters in the RBC membrane, including ion channels, shows that these cells are much more complex than expected.

2.1 Why one studies red blood cell membrane transport properties?

  • RBCs are highly specialised cells, present in all vertebrates (except some cold/ice-water fish (Ruud, 1954)).
  • Erythrocytes are produced in the bone marrow, differentiating from pluripotent cells during erythropoïesis.
  • A human RBC has a lifespan of around 120 days, before being removed from the circulation by macrophages, essentially in the spleen.
  • Patch Clamp Technique 172 RBCs have always occupied a primordial place in the investigations on membrane transport.
  • Moreover, the cellular structure and particularly that of mammalian of RBCs, has made them an ideal model for studying membrane transport.

2.2 The basis of red cell membrane permeability

  • The transport of the respiratory gases within the blood is highly dependent on electrolytes and the acid-base status of RBCs and they are strongly correlated with the permeability www.intechopen.com Patch-Clamp Analysis of Membrane Transport in Erythrocytes 173 properties of the membrane.
  • The authors will first present the different types and roles of membrane ion transporters that have been described in human RBCs, as they are the most studied among vertebrates.
  • The Na+/K+-ATPase maintains gradients for Na+ and K+, fuelling the secondary transporters present in the membrane.
  • Before the use of patch clamp, both components were frequently attributed to Band 3 protein activity, the electrogenic part resulting from either slippage in the exchange mechanism (Kaplan et al., 1983; Knauf et al., 1977), or tunneling (Frohlich, 1984; Knauf et al., 1983).
  • The emergence of patch clamp studies on red cell membranes (during the 80’s and 90’s for single channel, 2000’s for whole cell) first confirmed the presence of a calcium-activated potassium channel.

3.1.1 Size and deformability

  • The main problem when attempting to perform patch clamp on RBCs lies in their very small size.
  • The smallest RBCs are encountered in mammals and are enucleated (mean diameter: human 8µm, mammalian 2.1-9.4µm).
  • Nevertheless, deformability of the membrane remains a problem that could impair seal formation.
  • The main problem is to avoid entry of the cells into the pipette.
  • Their characteristics will be given in section 3.2.

3.1.2 Preparation

  • Blood is a non-fibrous connective tissue and collection and isolation of RBCs is therefore, a rather easy process.
  • This is a great advantage, meaning that to be patched these cells do not require any mechanic or enzymatic dissociation, and that they do not adhere to solid surface in vitro.
  • Blood must be drawn from human or animal with an anticoagulant (such as heparin or EDTA), and can be stored at 4°C for several days.
  • To isolate RBCs, simple centrifugation steps (usually four successive washings) at a speed of 3000 to 4000 rpm (around 2000g) are needed.
  • RBCs are the densest elements of blood, thus supernatant and buffy coat (white blood cells and platelets) can be discarded after each step.

3.2.1 Remarks on the use of patch clamp on small cells

  • When narrowing the tip of the pipette, its resistance Rpip is increased.
  • Work on an adapted shape of the pipette for RBCs (especially human RBCs) has led to the use of pipette with an Rpip between 10 and 15MΩ.
  • Up to 80% of seal attempts are successful with such pipettes, depending on the solutions used.
  • For whole-cell experiments, two resistances are in parallel in the electrical model: global membrane resistance Rm and seal resistance Rseal .
  • This can induce an underestimation of the channel conductance and a discrepancy between real and apparent reversal potentials in cell-attached experiments, and wrong global conductance and shift in I/V curves in whole-cell experiments.

Do these limitations apply to RBCs?

  • From suspension experiments, membrane resistance Rm of human RBCs was estimated in the range 1.106Ω.cm², with chloride resistance RCl ranging between 105 and 106 Ω.cm² (Hoffman et al., 1980).
  • This is in range of the seal values obtained on RBCs when using the patch clamp techniques.
  • Oxidized or malaria-infected RBCs, and cation conductance can be increased when cation channels are activated (with low external chloride concentrations for example).
  • Thus, for whole cell experiments, Rm remains much lower than Rseal and the current and voltage do not suffer high distortion.
  • The use of pipette immediately after pulling, their adapted shape, with rapidly tapering geometry, and the filtering of all pipette solutions with 0.2µm filters can maintain low Raccess values.

3.2.2 Electrodes, pipettes and seal

  • Ag wires are regularly anodised to maintain uniform oxidized coating.
  • When perfusion of bath solution is performed, it modifies junction potentials.
  • For that purpose, the JPCalc software developed by Peter Barry is very convenient (Barry, 1994).
  • The authors use thin borosilicate pipette with filament (this helps liquid filling) (GC150TF-10, Clark Electromedical Instruments), and pull them with a horizontal DMZ puller (Zeitz instruments, Germany).
  • The pipette capacitance can be measured and compensated, it is usually around 3pF.

3.2.3 Single channel recordings

  • The ion channels present in the RBC membrane can have a small unitary conductance, thus the most important task is to track and eliminate noise.
  • Efficient Faraday cages and link of all metallic elements (microscope, anti-vibration table, and micromanipulator) to the ground using low resistance cables are of primary importance.
  • In their studies, recordings are filtered with a low pass 3 kHz filter.
  • Before digitalisation, signal is displayed on an oscilloscope to monitor analogic signal live and continuously.
  • Then perfusion can modify the intracellular side solution and help characterising the channel activity.

3.2.4 Whole-cell recordings

  • After obtaining seal, records can be made for single channel studies.
  • This is achieved via imposition in the pipette of a brief electrical pulse (200ms, 500mV).
  • This rarely provokes damages to the seal.
  • Mammalian erythrocyte capacitance is estimated at 0.8 µF.cm-2 (Fettiplace et al., 1971), this gives a membrane capacitance around 1- 1.3 pF for red blood cells.
  • The nystatin-perforated patch clamp has also been used on human RBCs, especially to study cation channel activity.

4. The contributions of patch clamp techniques in describing human red blood cell membrane properties

  • The authors will list the various ion channels that have been described or suggested in the human RBC membrane, and list their main properties as well as recording conditions.
  • Their possible physiological role will be evoked, before a description of their implication in various pathological situations.

4.1.1 Gardos channel

  • As mentioned previously, the Gardos channel was the first channel described in RBCs membrane.
  • Until now, the Gardos channel remains the best characterised channel in human RBC membrane.
  • The resting free [Ca2+]i in an unstimulated cell is about 100nM, consequently Gardos channel activation in a physiological situation seems rare.

4.1.2 Non-selective cation channel

  • A voltage-activated cation channel in the human RBC membrane was originally proposed by Halperin (Halperin et al., 1989), and it was later described electrophysiologically using www.intechopen.com Patch Clamp Technique 182 single channel recordings (Christophersen & Bennekou, 1991).
  • It shows a hysteresis voltage dependence (Kaestner et al., 2000), that was shown using patch clamp as well as cell suspension potential measurements (Bennekou et al., 2004a).
  • During the last decade the group of Florian Lang has also described a voltage-independent cation channel in the human RBC membrane.
  • One cation channel seems to be identified as the Transient Receptor Potential Cation channel 6 (TRPC6), which fits with properties of the non-selective voltage-dependent cation channel.
  • Nevertheless, a few studies have given evidence that true Ca2+ channels might also be present in the RBCs membrane.

4.2 Anion channels

  • As described in the first part of this chapter, conductive anion permeability of the RBC membrane has long been exclusively attributed to band 3 via slippage, or tunneling mechanisms.
  • Nevertheless, various anion channel activities have been described essentially in the last decade, giving unambiguous evidence of the presence of anion channel in the human RBC membrane.
  • Knowledge has come partly from studies on Plasmodium-infected RBCs, showing spontaneous anion channel activity.
  • It is now known that these channels are endogenous proteins, upregulated in parasitized cells.
  • Nonetheless, anion channel inhibitors are generally poorly specific, and in cells lacking expression machinery, and where membrane majoritary proteins complicate proteomics studies, precise identification of these anion channels has been and still remains difficult.

4.2.1 Cystic Fibrosis Transmembrane conductance Regulator (CFTR)

  • The presence of CFTR protein in the human RBC membrane has long been debated.
  • Indeed, CFTR activity seems necessary for deformation-induced ATP release and the protein was detected using western blots (Abraham et al., 2001; Sprague et al., 1998).
  • Comparisons between RBCs from healthy donors and CF patients using whole-cell configuration allowed the description of a tiny current attributed to CFTR activity; but the role of CFTR remains unclear.
  • Indeed, its regulatory properties seem more important.
  • This was confirmed at the single channel level in non-infected human RBCs, with behaviour similar to a small anionic channels (around 10pS) in these two types of cells: its gating and kinetics were different, but the properties did not correspond to CFTR activity (Decherf et al., 2007).

4.2.2 Small conductance chloride channel / ClC-2

  • A small anion channel has been described in the human RBC membrane, and was designed as a ClC-2 channel.
  • In a subsequent study, the authors showed that this channel corresponds to the ClC-2 channel already described, thus being an endogenous channel activated upon infection (Bouyer et al., 2007).
  • Using the whole-cell configuration it was shown that this channel is activated by oxidation using 1mM tertbutylhydroperoxide (t-BHP) (Huber et al., 2004; Huber et al., 2002).
  • Zinc shows an inhibitory effect, with an IC50 around 100µM, whereas NPPB or furosemide are relatively ineffective (Shumilina & Huber, 2011).
  • This channel might contribute to the basal anionic conductance of human RBCs, clamping the cell potential to the chloride equilibrium potential.

4.2.3 Voltage-dependent anion channel / peripheral benzodiazepine receptor

  • Two recent works, using both cell-attached and whole-cell configurations have provided evidence for the presence of another type of anion channel in human RBC membrane.
  • This channel shows multiple conductance substates that are dependent on the presence of serum in the bath solution.
  • The identity of this channel remained undetermined until a second study by their group showed that a Voltage Dependent Anion Channel (VDAC) was present in the human RBC membrane (Bouyer et al., 2011a).
  • The TSPO component is considered to be primarily responsible for binding to PK 11195, while Ro5-4864 and other benzodiazepines may bind to all components of the PBR complex (Le Fur et al., 1983; McEnery et al., 1992).
  • The presence of such proteins in the RBC membrane raises many questions regarding their possible physiological role, but according to the properties of its components the authors can predict a major role in membrane transport, volume and redox status regulation, as well as cell differentiation and senescence.

4.3 Implication of RBC ion channels in pathophysiological situations

  • Their implication in various pathologies has been known for decades.
  • In particular, cation channel activity is a key factor of sickle cell disease pathology (Lew & Bookchin, 2005), as is anion channel activity in Plasmodium-infected human RBCs (Ginsburg et al., 1983; Kirk et al., 1994).

4.3.1 Sickle cell disease

  • In the sickle cell disease polymerisation of HbS haemoglobin under deoxygenated conditions leads to cell dehydration via efflux of potassium, chloride and osmotically obliged water.
  • Future research should focus on its identification.
  • This pathway was characterised using fluxes and haemolysis experiments during the 80’s and 90’s.
  • Electrophysiological description of the spontaneously active ion channels in infected RBCs membrane has been highly controversial, owing to the multiplicity of experimental conditions used by the different groups in the field.
  • It was also shown that supraphysiological ionic concentrations used in bath and pipette solutions modify anion channel activity: saturation of conductance and inhibition by lower open probability appeared beyond 0.6M of Cl- in solutions (Bouyer et al., 2007).

4.3.3 Senescence

  • During their circulatory life, RBCs tend to become progressively denser.
  • This correlates with a decline in Ca2+ pump activity that leads to KCl loss (via Gardos and anion channels) overcompensated by NaCl gain (Lew et al., 2007).
  • Activity of a non-selective cation channel has been linked to this phenomenon that tends to dissipate Na+ and K+ gradients late in the lifespan of RBCs.
  • This follows the same path: an external signal (oxidative stress, for example) triggers a rise in intracellular Ca2+, probably by activation of a non-selective cation channel by prostaglandin E2.
  • This provokes cell shrinkage, scramblase and calpain activation resulting in phosphatidylserine exposure and degradation of the cytoskeleton (reviewed in (Foller et al., 2008a; F. Lang et al., 2004; F. Lang et al., 2006)).

5. Comparative physiology: Use of patch-clamp in a evolutionary approach on vertebrates red cells

  • Comparative physiology of red blood cells membrane have been evident for many years regarding respiratory function and many studies highlighted the role of ion transporters in resting and challenging situations.
  • The use of the patch-clamp technique on models other than mammals are relatively limited.
  • This recovery is accomplished by selectively increasing the permeability of the plasma membrane during cell swelling to allow for efflux of specific intracellular osmolytes, thereby generating a driving force for water efflux.
  • Indeed, Agnathans (jawless vertebrates) are devoid of Cl-/HCO3- exchangers, but possess like other vertebrates a powerful anion conductance with low selectivity and which presents similar electrophysiological characteristics, as VDAC/PBR found in human RBCs (unpublished data).

6. Conclusion

  • Though having been explorated by various techniques during decades, the membrane permeability of RBCs is still not fully understood.
  • The use of patch-clamp techniques has proven to be very useful shedding light on a much more complicated situation than expected.
  • Indeed, RBCs are equipped with multiple transporters including various ion channels.
  • There is much evidence that RBCs plays a more complex role than simple oxygen supplier to tissues.
  • Then, the various ion channels in vertebrate RBCs could help describe a phylogeny of respiratory mechanisms throughout evolution, and lead to a better understanding of the role of ion channels in the human RBC membrane.

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9
Patch-Clamp Analysis of Membrane
Transport in Erythrocytes
Guillaume Bouyer, Serge Thomas and Stéphane Egée
Centre National de la Recherche Scientifique, Université Pierre et
Marie Curie Paris6, Station Biologique, Roscoff
France
1. Introduction
Among all the models used to study membrane transport, erythrocytes (Red Blood Cells,
RBCs) have probably been the most utilised cell type. Radioisotopes fluxes, isosmotic
haemolysis, ion content analysis (e.g. flame photometry), or fluorescence techniques have
been widely used to characterise the various transporters present in the RBCs membrane.
These techniques have allowed the description of several types of transporters such as
pumps, specific solute transporters, symporters or antiporters, and even ion channels.
However, the physiology of RBCs and their maintenance of homeostasis remains
incompletely understood, and electrophysiology has proven, since the first single-channel
recording on a human erythrocyte membrane thirty years ago, to be a very useful tool to
understand more deeply RBC membrane transport. Why does one use these techniques on a
small, non-excitable cell that has long been considered no more than an empty bag of
haemoglobin? The diversity of transporters in the RBC membrane, including ion channels,
shows that these cells are much more complex than expected. Indeed, ion channels now
described in the RBC membrane (from Mammals to other Vertebrates) are implicated in
important phenomena and functions throughout the cells lifespan (gas transports, cell
volume regulation, differentiation and death). In this chapter, we will describe the main
properties of the erythrocyte’s membrane transport system, how electrophysiological
techniques can be applied, and how they have contributed to the comprehension of
erythrocyte physiology with the description of the various ion channels that can be found in
RBC membrane.
2. Red blood cell membrane description
2.1 Why one studies red blood cell membrane transport properties?
RBCs are highly specialised cells, present in all vertebrates (except some cold/ice-water fish
(Ruud, 1954)). Their main role is the transport of respiratory gases, between tissues and lungs
or gills. Encapsulation of the respiratory pigment haemoglobin in a cell in vertebrates has
hugely increased the gas transport capacity of blood, and is a key point throughout evolution
of the animal kingdom. Erythrocytes are produced in the bone marrow, differentiating from
pluripotent cells during erythropoïesis. A human RBC has a lifespan of around 120 days,
before being removed from the circulation by macrophages, essentially in the spleen.
www.intechopen.com

Patch Clamp Technique
172
RBCs have always occupied a primordial place in the investigations on membrane
transport. First of all, even if their major role in respiration processes of vertebrates has been
known for long time, deciphering the precise role of the different membrane transporters
involved has been a long story. Indeed, membrane transport and especially ion permeability
are inseparable from the description of gas transport by erythrocytes. Oxygen diffuses freely
across RBC membranes, but its affinity to haemoglobin is highly dependant on cell
homeostasis and thus to transport regulation across cell membrane. Moreover, the high
carbon dioxide transport capacity of blood is essentially supported by the Jacobs-Stewart
cycle between red cells and plasma, relying on the existence of specialised ion transporters
in the erythrocyte membrane (Jacobs & Stewart, 1942).
Moreover, the cellular structure and particularly that of mammalian of RBCs, has made
them an ideal model for studying membrane transport. Mature mammalian erythrocytes are
devoid of intracellular organites, and this means that they consist of a single compartment,
simplifying many approaches for transport studies. During the end of cellular
differentiation, the nucleus is extruded from the normoblats and engulfed by surrounding
macrophages (Yoshida et al., 2005), and the other organelles are removed during the
maturation of reticulocytes into erythrocytes, probably mainly via autophagy (Kundu et al.,
2008; Mortensen et al., 2010). This makes mature erythrocytes from mammals a very easy-to-
use model for plasma membrane transport studies: intracellular constant measurements (ion
or metabolite concentrations, pH) and flux experiments are easier than in any other type of
cell containing multiple compartments.
Finally, another reason for studying red blood cell membrane transporters is the nature of
blood, as a non-fibrous connective tissue : the fact that these cells are naturally in suspension
and thus do not need any mechanical, enzymatical or chemical treatment before use in any
kind of experiment also makes them easy to handle. Moreover, apart from ethical questions,
it is always technically easy to draw blood from animals or humans, and purification of
RBCs from blood only requires few centrifugation steps.
Thus, many scientists have used RBCs throughout history to describe the diversity of
transporters in the plasma membrane, and to understand their role in the maintenance of
homeostasis. Among all these studies, several have revealed essential characteristics of cell
membrane permeability. In particular, as early as 1960 work on RBCs allowed Tosteson and
Hoffman to complete the description of the “pump and leak” steady-state concept using
sheep RBCs (Tosteson & Hoffman, 1960). In 1966, Schatzmann described for the first time an
ATP-fuelled Ca
2+
pump and this discovery was made using human erythrocytes
(Schatzmann, 1966). Furthermore, as early as 1981 these cells were among the first using the
patch-clamp technique that provided direct electrophysiological evidence for the presence
of ionic channels in the plasma membrane (first recordings of a K
Ca
channel by Owen
Hamill) (Hamill, 1981). They were also the cells in which aquaporins were first described
and for which Peter Agre won the Nobel Prize (Agre et al., 1993). Nowadays, many studies
on the properties of RBC membrane transport are made either in physiological or
pathophysiological situations, and the patch clamp technique has become an essential tool
in their characterisation and comprehension.
2.2 The basis of red cell membrane permeability
The transport of the respiratory gases within the blood is highly dependent on electrolytes
and the acid-base status of RBCs and they are strongly correlated with the permeability
www.intechopen.com

Patch-Clamp Analysis of Membrane Transport in Erythrocytes
173
properties of the membrane. Indeed, the erythrocyte membrane is endowed with a variety
of membrane transporters, whose role is absolutely vital to maintain cell homeostasis. In this
section, we will first present the different types and roles of membrane ion transporters that
have been described in human RBCs, as they are the most studied among vertebrates.
The main characteristics of human RBC membrane ion permeability are linked to the
unusual composition of these cells. The encapsulation of ~ 5 mmol of impermeable
haemoglobin per litre of intracellular water in a cell moving in a plasma environment, that
has a much lower protein concentration, creates a huge osmotic pressure. As explicitly
formulated in the ‘pump-leak’ concept (Tosteson & Hoffman, 1960), the risk of
colloidosmotic swelling and bursting is prevented by a very low membrane permeability to
cations, allowing the pumps Na
+
/K
+
-ATPase and Ca
2+
-ATPase to extrude the residual Na
+
and Ca
2+
leaks at minimal metabolic cost. The red cell Ca
2+
-ATPase is so powerful that it
maintains intracellular concentration below micromolar concentrations (Lew et al., 1982;
Schatzmann, 1983). The Na
+
/K
+
-ATPase maintains gradients for Na
+
and K
+
, fuelling the
secondary transporters present in the membrane. Indeed, a potassium/chloride
cotransporter (KCC, identified as KCC1 (C.M. Pellegrino et al., 1998)), a
potassium/sodium/two chloride cotransporter (NKCC) (Haas, 1989) and a sodium/proton
(Na
+
/H
+
) exchanger (Semplicini et al., 1989) have been described in the RBC membrane.
By contrast, the RBC membrane is characterised by a huge anion permeability that is
essentially linked to the respiratory function: a million copies per cell of electroneutral Cl
-
/HCO
3
-
exchanger (called Band 3) permit 85% of the CO
2
produced in the tissues to be
transported in the blood as HCO
3
-
ions, via the Jacobs/Stewart cycle (Figure 1). This protein
was identified in 1972 by Cabantchik and Rothstein (Cabantchik & Rothstein, 1972), even if
RBC anion permeability had been studied for long time.
Fig. 1. The Jacobs/Stewart cycle in tissues. In lungs, cycle goes the other way.
It was known for long that RBCs anion permeability could be divided into two components:
a large exchange component fundamental for the CO
2
-carrying capacity of the blood (Gunn
et al., 1973), and a much smaller electrogenic component that normally determines the RBC
resting potential (Hunter, 1977; Lassen et al., 1978). This conductive part of chloride
permeability ensures a dissipation of chloride gradient across red cell membrane: the
membrane potential is clamped at the equilibrium potential for chloride (-12mV) ensuring
Medium Cell
CO
2
CO
2
HCO
3
-
Cl
-
H
2
O
H
+
Carbonic
Anh
y
drase
Band 3
HCO
3
-
Cl
-
www.intechopen.com

Patch Clamp Technique
174
that Band 3 never has to fight against a chloride gradient to transport bicarbonate ions
across red cell membrane. Before the use of patch clamp, both components were frequently
attributed to Band 3 protein activity, the electrogenic part resulting from either slippage in
the exchange mechanism (Kaplan et al., 1983; Knauf et al., 1977), or tunneling (Frohlich,
1984; Knauf et al., 1983).
Besides these cotransports, a single conductive pathway had been described in RBCs before
the appearance of patch clamp techniques: Gardos had shown in the 50’s that an
electrogenic, calcium-dependent potassium pathway was present in the human red cell
membrane. This has since become known as the Gardos effect, linked to the activity of a
calcium-activated potassium channel (Gardos, 1956, 1958).
For a long time, this list of membrane transporters was assumed to be complete: Band 3
mediated the very high anion permeability of red cells (via anion exchange and a much
smaller electrogenic gating), and the tiny cationic permeability was supported by various
powerful pumps and cation transporters carrying out homeostasis maintenance. The
emergence of patch clamp studies on red cell membranes (during the 80’s and 90’s for single
channel, 2000’s for whole cell) first confirmed the presence of a calcium-activated potassium
channel. It was described by single channel recordings by Hamill in 1981 (Hamill, 1981), and
identified later as a member of the K
Ca
channel family (hSK4, now called Gardos channel)
(Hoffman et al., 2003). But these patch clamp studies, first using single channel (cell-attached
and excised inside-out) and then whole-cell configurations, also brought evidence of a more
complex situation than expected. The groups in the fields showed the existence of various
anion and cation channels in the red cell membrane from different vertebrate species, and
even if their role in physiological situations still remains poorly understood, their
implication in several red cell pathologies is unambiguous and considerable.
It has taken much effort to first apply and then adapt patch clamp techniques to these tiny,
non excitable cells; especially with the previous models of red cell membrane permeability
that did not predicted the presence of various ion channels. Indeed, description of these
different channel types in RBCs, using patch clamp techniques, has raised many questions:
how do they interfere with the regulation of intracellular homeostasis, cell differentiation or
death? But their presence also fits with a new vision of red cells, being much more than an
empty bag of haemoglobin, notably regarding the recently discovered role of red cells in
vascular tone regulation (Sprague et al., 2007).
3. Technical specificities of red blood cells regarding the patch-clamp
technique
The technical aspects described here will focus on human red blood cells, as they are more
described and studied than erythrocytes from any other species.
3.1 Red blood cell specificities
3.1.1 Size and deformability
The main problem when attempting to perform patch clamp on RBCs lies in their very small
size. The smallest RBCs are encountered in mammals and are enucleated (mean diameter:
human 8µm, mammalian 2.1-9.4µm). They are nucleated and slightly bigger in other
www.intechopen.com

Citations
More filters
Journal ArticleDOI
TL;DR: The current knowledge on RBC deformability in different forms of hereditary hemolytic anemia is reviewed and secondary mechanisms involved in R BC deformability are described.
Abstract: Deformability is an essential feature of blood cells (RBCs) that enables them to travel through even the smallest capillaries of the human body. Deformability is a function of (i) structural elements of cytoskeletal proteins, (ii) processes controlling intracellular ion and water handling and (iii) membrane surface-to-volume ratio. All these factors may be altered in various forms of hereditary hemolytic anemia, such as sickle cell disease, thalassemia, hereditary spherocytosis and hereditary xerocytosis. Although mutations are known as the primary causes of these congenital anemias, little is known about the resulting secondary processes that affect RBC deformability (such as secondary changes in RBC hydration, membrane protein phosphorylation, and RBC vesiculation). These secondary processes could, however, play an important role in the premature removal of the aberrant RBCs by the spleen. Altered RBC deformability could contribute to disease pathophysiology in various disorders of the RBC. Here we review the current knowledge on RBC deformability in different forms of hereditary hemolytic anemia and describe secondary mechanisms involved in RBC deformability.

195 citations

Book ChapterDOI
TL;DR: The Gárdos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, CaV2.1, a Ca2+-inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance Psickle are related.
Abstract: Free Calcium (Ca2+) is an important and universal signalling entity in all cells, red blood cells included. Although mature mammalian red blood cells are believed to not contain organelles as Ca2+ stores such as the endoplasmic reticulum or mitochondria, a 20,000-fold gradient based on a intracellular Ca2+ concentration of approximately 60 nM vs. an extracellular concentration of 1.2 mM makes Ca2+-permeable channels a major signalling tool of red blood cells. However, the internal Ca2+ concentration is tightly controlled, regulated and maintained primarily by the Ca2+ pumps PMCA1 and PMCA4. Within the last two decades it became evident that an increased intracellular Ca2+ is associated with red blood cell clearance in the spleen and promotes red blood cell aggregability and clot formation. In contrast to this rather uncontrolled deadly Ca2+ signals only recently it became evident, that a temporal increase in intracellular Ca2+ can also have positive effects such as the modulation of the red blood cells O2 binding properties or even be vital for brief transient cellular volume adaptation when passing constrictions like small capillaries or slits in the spleen. Here we give an overview of Ca2+ channels and Ca2+-regulated channels in red blood cells, namely the Gardos channel, the non-selective voltage dependent cation channel, Piezo1, the NMDA receptor, VDAC, TRPC channels, CaV2.1, a Ca2+-inhibited channel novel to red blood cells and i.a. relate these channels to the molecular unknown sickle cell disease conductance Psickle. Particular attention is given to correlation of functional measurements with molecular entities as well as the physiological and pathophysiological function of these channels. This view is in constant progress and in particular the understanding of the interaction of several ion channels in a physiological context just started. This includes on the one hand channelopathies, where a mutation of the ion channel is the direct cause of the disease, like Hereditary Xerocytosis and the Gardos Channelopathy. On the other hand it applies to red blood cell related diseases where an altered channel activity is a secondary effect like in sickle cell disease or thalassemia. Also these secondary effects should receive medical and pharmacologic attention because they can be crucial when it comes to the life-threatening symptoms of the disease.

41 citations

Journal ArticleDOI
TL;DR: The findings suggest that MPO functions as a mediator of novel regulatory mechanism in microcirculation, indicating the influence of MPO-induced abnormalities on RBC deformability under pathological stress conditions.

29 citations

Journal ArticleDOI

25 citations


Cites background from "Patch-Clamp Analysis of Membrane Tr..."

  • ...At present, the molecular identity of this particular channel remains unknown (Kaestner, 2011; Bouyer et al., 2012), and it has alternatively been proposed to reflect a conductance state of the voltage-dependent anion channel (VDAC) (Bouyer et al....

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  • ...At present, the molecular identity of this particular channel remains unknown (Kaestner, 2011; Bouyer et al., 2012), and it has alternatively been proposed to reflect a conductance state of the voltage-dependent anion channel (VDAC) (Bouyer et al., 2011)....

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Journal ArticleDOI
TL;DR: The Gardos channel, named after an effect initially described by George Gardos based on flux-experiments, was the first channel shown in human RBCs by patch-clamp using single channel recordings and is in the RBC research field sometimes presented in a rather fragmentary way.
Abstract: Both patch-clamp and molecular biology provide powerful tools to investigate ion channels. However, the approaches couldn't be more different. While patch-clamp probes protein function on the scale of a single cell or even down to a single molecule, molecular biology includes techniques that identify the protein, and requires mostly cell populations. According to current knowledge and compared to other cell types, red blood cells (RBCs) possess a rather small variety of ion channels. Nevertheless, both techniques are still revealing new channels. The challenge is to keep the results of both methods in agreement. Such consistency can be shown for a number of channels; just to name successful examples, the voltage-dependent anion channel (VDAC) (Bouyer et al., 2011, 2012) and the NMDA-receptor (Makhro et al., 2013; Hanggi et al., 2014). However, providing a general correlation between patch-clamp and molecular biology derived results has had limited success (Kaestner, 2011; Bouyer et al., 2012). One reason for this difficulty is that techniques themselves or their particular application have certain shortcomings. Patch-clamp, although an extremely powerful technique to investigate RBCs (Hamill, 1983), is in the RBC research field sometimes presented in a rather fragmentary way. An example is a channel, published in 2000, that was supposed to be induced in RBCs by the malaria parasite (Figure 2 in Desai et al., 2000). In contrast, a channel with very similar properties had already been described in healthy RBCs in 1989 (Figure 6 in Schwarz et al., 1989). After years of debate (for examples, see Egee et al., 2002; Huber et al., 2002; Staines et al., 2003, 2007), it became accepted that the increased conductance in malaria infected RBCs was mediated by endogenous ion channels of RBCs (Bouyer et al., 2007). For molecular biology-based investigations, RBCs impose a twofold challenge: Most genetic approaches are limited to precursor cells, because mammalian RBCs contain neither a nucleus nor ribosomes as a translational machinery. It appears to be extremely difficult to isolate pure preparations of RBCs (Minetti et al., 2013). Even in cell preparations filtered on cellulose (Beutler et al., 1976), RNA of tyrosine phosphatase (CD45—a marker for non-RBCs) was found in next generation sequencing. Only further fluorescence-activated RBC sorting revealed CD45-free preparations. Therefore, proofs for the molecular identity of ion channels in RBCs often include indirect methods. For example, the Gardos channel, named after an effect initially described by George Gardos based on flux-experiments (Gardos, 1958), was the first channel shown in human RBCs by patch-clamp using single channel recordings (Hamill, 1981). The Gardos channel was later identified to be encoded by the KCNN4 gene (KCa3.1protein, also called hSK4 channel) (Hoffman et al., 2003). Although the RT-PCR of reticulocytes and the Western blots of RBCs look convincing one needs to consider point (ii) above. All other arguments such as Northern blots of human erythroid progenitor cells or properties of heterologously expressed KCNN4 vs. KCNN3 (Hoffman et al., 2003) are indirect in nature. On the patch-clamp side one can find numerous single channel recordings of the Gardos channel (Hamill, 1981, 1983; Grygorczyk et al., 1984; Schwarz et al., 1989), but whole cell recordings are almost missing. Some electrophysiologists even believe the Gardos channel is unmeasurable in the whole-cell configuration of RBCs. I am only aware of one paper in which the authors were courageous enough to publish whole-cell recordings of the Gardos channel in human RBCs (Kucherenko et al., 2013). The lack of more attempts is not surprising considering the estimation of the number of channels per cell based on single channel recordings at approximately 10 (Grygorczyk et al., 1984), which renders whole-cell recordings difficult. Another example is Piezo1—this mechano-sensitive channel was only recently discovered (Coste et al., 2010) and associated with the anemic disease hereditary xerocytosis (HX) due to mutations of Piezo1 found in HX patients (Zarychanski et al., 2012). Pharmacological modulations in patch-clamp experiments suggest that Piezo1 may also contribute to Psickle in sickle cell disease RBCs (Bae et al., 2011; Ma et al., 2012; Gallagher, 2013). Beside all these findings and a biophysical characterisation of Piezo1 in heterologous expression systems (Bae et al., 2011; Gottlieb and Sachs, 2012), the channels' direct functional or molecular proof in human RBCs remains rather elusive—patch-clamp recordings in HX RBCs lack (statistical) comparison in healthy controls (Figure 2 in Archer et al., 2014). Although Piezo1 abundance and function in mouse RBCs has been shown (Cahalan et al., 2015), so far I have not seen any convincing Piezo1 protein data, such as immunocytochemistry or Western blots, based on human RBCs. However, indirect evidence, e.g., measurements of Gardos channel activity induced by membrane deformations (Dyrda et al., 2010), where deformations are likely to activate Piezo1 eventually triggering Gardos channel activity, contribute to the overall picture. The intention to present these prominent examples is not to doubt the existence of the Gardos channel or the Piezo1 in RBCs, but to illustrate the difficulties in revealing channel identities or saying it with other words: bringing electrophysiology and molecular biology into agreement. To achieve this goal it seems compulsory to consider some points when investigating ion channels in RBCs: Functional studies are always most convincing. Beside patch-clamp recordings those include fluorescence-based methods and tracer flux experiments. Conviction increases if it can be proved that effects originate exclusively from RBCs [see point (ii) above]. This condition is relatively easily met when experiments are performed on single cells under visual inspection, such as patch-clamp or fluorescence imaging. Cell population measurements require purification efforts. Centrifugation based methods are insufficient (Minetti et al., 2013). Additional filtering, e.g. through cellulose (Beutler et al., 1976), improves the situation. Filtering should be followed by a gelatine zymography (Achilli et al., 2011), which works fine for human RBCs but is insufficient for mouse RBCs. Quantification through assays detecting tyrosine phosphatase is even better. For really pure RBC preparations high quality sorting procedures based on CD45 antibodies should be implemented. Patch-clamp recordings require a full characterisation based on ion selectivity and other biophysical or pharmacological parameters. Channel identification should not preferentially rely on the appearance of traces or the general compatibility of the trace with the hypothesis (e.g, Kaestner and Bernhardt, 2002; Locovei et al., 2006; Archer et al., 2014). Although sometimes one gets the impression that when RBCs are concerned, molecular biology competes with patch-clamp as indicated by the title, it is obvious and necessary that both techniques need to synergistically complement each other to effectively reveal the complexity of RBCs.

18 citations


Cites background from "Patch-Clamp Analysis of Membrane Tr..."

  • ...However, providing a general correlation between patch-clamp and molecular biology derived results has had limited success (Kaestner, 2011; Bouyer et al., 2012)....

    [...]

  • ...Such consistency can be shown for a number of channels; just to name successful examples, the voltage-dependent anion channel (VDAC) (Bouyer et al., 2011, 2012) and the NMDA-receptor (Makhro et al., 2013; Hänggi et al., 2014)....

    [...]

References
More filters
Journal ArticleDOI
TL;DR: Current knowledge regarding the molecular identity of these transport pathways and their regulation by, e.g., membrane deformation, ionic strength, Ca(2+), protein kinases and phosphatases, cytoskeletal elements, GTP binding proteins, lipid mediators, and reactive oxygen species are reviewed.
Abstract: The ability to control cell volume is pivotal for cell function. Cell volume perturbation elicits a wide array of signaling events, leading to protective (e.g., cytoskeletal rearrangement) and adap...

1,239 citations


"Patch-Clamp Analysis of Membrane Tr..." refers background in this paper

  • ...Most vertebrate cells lose K+ and Cl− during RVD (Hoffmann et al., 2009)....

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Journal ArticleDOI
TL;DR: The mitochondrial benzodiazepine receptor has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized, finding that VDAC and ADC, outer and inner mitochondrial membrane channel proteins, respectively, together with the 18-kDa subunit, may comprise mBzR at functionally important transport sites at the junction of two mitochondrial membranes.
Abstract: The mitochondrial benzodiazepine receptor (mBzR) has been solubilized with retention of reversible ligand binding, and the associated subunits were characterized. mBzR comprises immunologically distinct protein subunits of 18-, 30-, and 32-kDa. The 18-kDa protein is labeled by the isoquinoline carboxamide mBzR ligand [3H]PK14105, whereas the 30- and 32-kDa subunits are labeled by the benzodiazepine (Bz) ligands [3H]flunitrazepam and [3H]AHN-086. Selective antibodies and reagents identify the 32- and 30-kDa proteins as the voltage-dependent anion channel (VDAC) and the adenine nucleotide carrier (ADC), respectively. While isoquinoline carboxamide and Bz ligands target different subunits, they interact allosterically, as the binding of Bz and isoquinoline carboxamide ligands is mutually competitive at low nanomolar concentrations. Moreover, eosin-5-maleimide and mercuric chloride inhibit [3H]PK11195 binding to the intact receptor via sulfhydryl groups that are present in ADC. VDAC and ADC, outer and inner mitochondrial membrane channel proteins, respectively, together with the 18-kDa subunit, may comprise mBzR at functionally important transport sites at the junction of two mitochondrial membranes.

703 citations


"Patch-Clamp Analysis of Membrane Tr..." refers background in this paper

  • ...The TSPO component is considered to be primarily responsible for binding to PK 11195, while Ro5-4864 and other benzodiazepines may bind to all components of the PBR complex (Le Fur et al., 1983; McEnery et al., 1992)....

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Journal ArticleDOI
TL;DR: The aim of this review is to outline the contribution of such junction potentials and the errors resulting from measurements on small cells and to indicate how adequate junction potential corrections can be applied and the true values of underlying membrane parameters determined for such cells.
Abstract: Since the early 1980s, the patch-clamp technique (Hamill et al., 1981) has been of particular value in investigating the properties of ion channels in cells. When used in either the intact or excised configurations, the properties of individual ionic channels can be directly measured. In addition, the whole-cell configuration can be used to investigate the total response of the full complement of channels in a cell. The whole-cell configuration is of particular value in exploring the properties of very small cells which are not readily accessible to conventional microelectrode techniques. in all of the above measurements, there are two potential sources of error, in every situation there may be significant errors due to uncompensated junction potentials, which m a y a p p e a r to be eliminated by the normal zeroing procedure whereby residual potentials between pipette and bath solutions are offset prior to patch formation. In addition, in the intact and whole-cell patch configurations, the effect of the cells being small can introduce radical errors in the measurement of single-channel and whole-cell properties. The aim of this review is firstly to outline the contribution of such junction potentials and the errors resulting from measurements on small cells and secondly to indicate how adequate junction potential corrections can be applied and the true values of underlying membrane parameters determined for such cells. Where necessary, appropriate equations have been presented. Much of the material is a review of published work. However, the review also seeks to extend the implications of that work and, in particular, it also includes (in an appendix) a timedependent solution of the current relaxation following a channel closure.

672 citations


"Patch-Clamp Analysis of Membrane Tr..." refers background in this paper

  • ...Barry and Lynch conclude their work (Barry & Lynch, 1991) by the equation (1) revealing the distortion between the apparent and real conductance of the single channel conductance : γc=γapp(1+Rm/Rpatch) (1) where γc and γapp are the real and apparent conductance of the channel, respectively....

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  • ...However, as suggested by Barry and Lynch (Barry & Lynch, 1991), distortion of the potential really applied to the membrane can happen in small cells, in relation with the global membrane resistance of the cell Rm....

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Journal ArticleDOI
TL;DR: Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation ofCHIP function may provide novel treatments of renal failure and other clinical problems.
Abstract: Despite longstanding interest by nephrologists and physiologists, the molecular identities of membrane water channels remained elusive until recognition of CHIP, a 28-kDa channel-forming integral membrane protein from human red blood cells originally referred to as "CHIP28." CHIP functions as an osmotically driven, water-selective pore; 1) expression of CHIP conferred Xenopus oocytes with markedly increased osmotic water permeability but did not allow transmembrane passage of ions or other small molecules; 2) reconstitution of highly purified CHIP into proteoliposomes permitted determination of the unit water permeability, i.e., 3.9 x 10(9) water molecules.channel subunit-1 x s-1. Although CHIP exists as a homotetramer in the native red blood cell membrane, site-directed mutagenesis studies suggested that each subunit contains an individually functional pore that may be reversibly occluded by mercurial inhibitors reacting with cysteine-189. CHIP is a major component of both apical and basolateral membranes of water-permeable segments of the nephron, where it facilitates transcellular water flow during reabsorption of glomerular filtrate. CHIP is also abundant in certain other absorptive or secretory epithelia, including choroid plexus, ciliary body of the eye, hepatobiliary ductules, gall bladder, and capillary endothelia. Distinct patterns of CHIP expression occur at these sites during fetal development and maturity. Similar proteins from other mammalian tissues and plants were later shown to transport water, and the group is now referred to as the "aquaporins." Recognition of CHIP has provided molecular insight into the biological phenomenon of osmotic water movement, and it is hoped that pharmacological modulation of CHIP function may provide novel treatments of renal failure and other clinical problems.

655 citations


"Patch-Clamp Analysis of Membrane Tr..." refers background in this paper

  • ...They were also the cells in which aquaporins were first described and for which Peter Agre won the Nobel Prize (Agre et al., 1993)....

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Journal ArticleDOI
TL;DR: The JPCalc program has been designed to graphically illustrate how junction potential contributions arise in various electrophysiological situations, to enable the magnitude and direction of those values to be readily calculated and to show clearly how the resultant appropriate corrections need to be applied to experimental measurements.

645 citations


"Patch-Clamp Analysis of Membrane Tr..." refers methods in this paper

  • ...For that purpose, the JPCalc software developed by Peter Barry is very convenient (Barry, 1994)....

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Frequently Asked Questions (2)
Q1. What have the authors stated for future works in "Patch-clamp analysis of membrane transport in erythrocytes" ?

Finally, the characterisation of the various ion channels in diverse vertebrate species is of high interest and constitutes an important field for future research. 

In this paper, the authors describe the main properties of the erythrocyte 's membrane transport system, how electrophysiological techniques can be applied, and how they have contributed to the comprehension of the various ion channels that can be found in RBC membrane. 

Trending Questions (1)
What type of transporters are expressed on erythrocytes?

The erythrocyte membrane is endowed with a variety of membrane transporters, including pumps, specific solute transporters, symporters or antiporters, and ion channels.