The Hematology Journal (2001) 2, 200 ± 205
ã 2001 The European Haematology Association All rights reserved 1466 ± 4680/01 $15.00
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HYPOTHESIS AND DEBATE
How do sickle cells become dehydrated?
Patrick Merciris1 and FrancËoise Giraud*,1
1
Laboratoire des Biomembranes et Messagers Cellulaires, Universite Paris XI-Orsay, France
The Hematology Journal (2001) 2, 200 ± 205
Keywords: sickle cells; hemoglobin F; K/Cl cotransport; KCa channels; serine/threonine phosphatase
2A; protein tyrosine kinase
Introduction
In sickle cell anemia, dehydrated erythrocytes (SS
RBC) are involved in the propagation of the vaso-
occlusive process, which results in the most severe
complication in sickle cell disease (e.g. stroke).
Deoxygenation leads to red cell dehydration, and
hence increases the rate of hemoglobin S (HbS)
polymer formation, which subsequently sickles the
cell and retards RBC transit through the microvascu-
lature. Dehydrated SS RBC exhibit decreased filter-
ability and poor deformability, contributing to acute
and chronic vaso-occlusive episodes and organ damage
and to early removal from the circulation, uncompen-
sated by erythropoiesis, resulting in hemolytic anemia.1
The hydration state and the hemoglobin F (HbF)
content of RBC are known to in¯uence their in vivo
survival: SS RBC, and especially denser fractions, have
a much shorter lifespan than normal RBC; and, the
lifespan of cells with high levels of HbF (F cells),
particularly in SS patients, is six to eight weeks,
whereas that of non-F cells is only about two weeks.2
Populations of SS RBC are highly heterogeneous
with respect to age, density, HbF and cation contents
(Figure 1). The densest SS cells are, on average,
younger than lesser dense cells, and, in vivo,
subpopulations of young cells dehydrate at di€erent
rates,3 with a fraction (mostly reticulocytes) being
susceptible to rapid dehydration after deoxygenation
in vitro. Dehydration is the result of a cationic loss,
followed by Cl7 and water leaks to maintain
electroneutrality and osmolarity of the cell. Deoxy-
genation-induced sickling leads to a permeabilization
of the SS RBC membrane for mono and divalent
cations, resulting in a net movement of Na+, K+, Mg2+
and Ca2+ along their electrochemical gradients, a
*Correspondence: F Giraud, Biomembranes et Messagers Cellulaires,
CNRS UMR 8619, Bat 440, Universite Paris XI 91405 Orsay Cedex,
France;
Tel: +33 1 69157644; Fax: +33 169154961
E-mail: francoise.giraud@ibaic.u-psud.fr
Received 13 November 2000; accepted 25 January 2001
process named sickling-induced pathway (SIP).4 Stimu-
lation of SIP leads to initially balanced Na+ and K+
¯uxes, but by increasing the Na+i/K+i ratio, this
stimulation results in the activation of the Na/K
pump and slow dehydration. The contribution of this
mechanism is considered to be minor and cannot
account for the extreme K+ depletion of highly dense
cells.1
Two K+ transporters have a high capacity for
mediating rapid K+ loss and dehydration in RBC:
the Ca2+-activated K channels (KCa channels) and the
K/Cl cotransport (KCl-cot). KCa channels, if maximally
activated, dehydrate normal RBC very rapidly. They
are activated in deoxygenated SS RBC, following the
stimulation of Ca2+ in¯ux by SIP.3,5 However, as HbF
delays and decreases HbS polymerization and hence
reduces SIP, KCa-channel activation will exclude most
of the F cells.3 KCl-cot is highly active in SS RBC, and
particularly in young RBC, where it can promote a
rapid dehydration when activated by cell swelling or
acid pH.6 Until recently, it was thought that KCl-cot is
inhibited by deoxygenation.7,8
Hypotheses and controversies on sickling-induced
pathway
The nature of SIP and whether it corresponds to the
activation of a pre-existing system or to a newly
formed pathway are presently unknown. As SIP is
sustained during deoxygenation, reversed on re-
oxygenation and prevented in F cells,3,9 a ®rst
hypothesis would suggest a relation to HbS polymer-
ization and morphological sickling.10 The properties of
SIP (lack of selectivity for cations, dependence on
membrane potential, but insensitivity to inhibitors of
all the known cationic transporters) are those of a
di€usible pathway or a channel with low selectivity
among metal cations.4 Na+ in¯ux mediated by SIP is
reduced by external Ca2+,3,11 suggesting a competition
between the two cations for the same transporter.

Figure 1 Heterogeneity of density-fractionated SS RBC. SS RBC
were separated in 4 or 3 density fractions, with de®ned density
limits, by centrifugation on a density gradient preparation (A, B)
or in 3 fractions corresponding to the top (low density), the
middle (intermediate density) or the bottom (high density) of the
density gradient preparation (C), as indicated in the inset in each
panel. (A) Distribution of the cells and mean corpuscular Hb
concentration (MCHC), percentage of reticulocytes, of irrever-
sibly sickled cells (ISC) and of HbF in each fraction; LD, low
density cells; ND, normal density cells; ID, intermediate density
cells; HD, high density cells.20 (B) Distribution of the cells and
percentage of F-RBC, of T+ cells and of T+F+ cells in each
fraction.19 (C) Concentration of Na+, K+ and Mg2+ in top,
middle and bottom fraction.3,6,25
Some inhibitors of band 3 anion transport, such as
4,4'-diisothiocyano-2,2' disulfostilbene (DIDS), can
reduce SIP, but with a concentration dependence
di€ering from that of anion transport.4,12 Moreover,
DIDS e€ect on SIP occurs without inhibiting
morphological sickling. Therefore, if there is a
relationship between sickling and SIP, it would involve
di€erent steps, with DIDS inhibiting one of them. The
interaction between HbS and the cytoplasmic domain
of band 3 might provide a site that is energetically
favorable for nucleation,9 and bundles of adjacent
Hypothesis: sickle cell dehydration
P Merciris and F Giraud
201
polymers would therefore generate oligomeric aggre-
gates of band 3, functioning as a large ion channel. In
favor of this model, deoxy (and denatured) Hb have a
much higher anity for band 3 than oxyHb, and
clusters of band 3 are found to be locally associated
with membrane-bound Heinz bodies in aged and SS
RBC. However, to be valid such a mechanism would
require that oligomerization of band 3 is suciently
rapid to precede the initiation of the cationic
permeabilization. In fact, autologous IgG binding on
the surface of SS RBC ± which re¯ects the formation
of band 3 clusters ± is a slow process, which becomes
signi®cant only after at least 16 h of deoxygenation,13 a
time scale incompatible with the rapid activation of
SIP.
A second hypothesis implies that the protrusion of
spicule HbS polymers, which causes uncoupling of the
membrane from the skeleton,14 would exert a distortion
on the membrane, leading to the activation of non-
selective mechano-sensitive, or stretch-activated, cation
channels.11 Cation permeability, stimulated by stress-
induced deformation of normal RBC, exhibits many of
the characteristics of SIP: in¯ux of Ca2+ and activation
of KCa channels, balanced movements of Na+ and K+
that are inhibited by DIDS; and inhibition of Na+
in¯ux by external Ca2+.15,16 However, SIP and
deformation-induced ¯uxes are unlikely to be
mediated by the same mechanism, as the concentra-
tion dependence of DIDS inhibition is distinct for the
two processes. In addition, stress-induced cation
transport is more consistent with a pathway formed
by rare ¯uctuations in the membrane structure than
with the activation of mechano-sensitive channels.16
Isolated spicule vesicles of deoxygenated SS cells
contain non-selective cation channels, with character-
istics similar to SIP, but how they are activated
remains to be determined.17
KCl-cot vs KCa-channel activation: what is the
initial trigger for dehydration?
The e€ect of deoxygenation on the behavior of a very
young and age-de®ned population of SS cells, which
still contain transferrin receptors (T+) and either HbF
(T+F+) or no HbF (T+F7) has been analysed.18,19 T+
cells dehydrate more than T7 cells with either 4 h
continuous deoxygenation or oxy-deoxy (O-D) cycles.
Under the former conditions, dehydration proceeds by
activation of KCa channels, while under the latter
conditions, both KCl-cot and KCa channels are
activated. Acid stimulation (pH 7.2) of KCl-cot in
moderately dense T+F+ or T+F7 cells promotes, to a
similar extent, the formation of intermediate density
T+ cells, consistent with a ®rst, HbF-independent,
dehydration stage. However, as the circulating densest
T+ cells (41.094) contained little or no HbF (Figure
1B), and have more KCl-cot activity (measured after
rehydration and low pH activation) than those which
remain light, the second stage of dehydration of these
young cells, from moderately dense to hyperdense, is
The Hematology Journal
 Hypothesis: sickle cell dehydration
P Merciris and F Giraud
202
considered to be sickling and HbF-dependent and
therefore involves KCa-channel activation.18,19
A fraction of cells with normal density (ND; 1.08 ±
1.09 g/mL), enriched in reticulocytes (Figure 1A),
generates dense cells of intermediate density (ID;
1.09 ± 1.114 g/mL) and hyperdense (HD; 41.114 g/
mL) cells by independent mechanisms,20 when exposed
for 22 h to O-D sickling at pH 6.8, in the presence of
Ca2+. Incubations in Cl7, NO37 or EGTA media or
with speci®c inhibitors, provide evidence for the role of
KCl-cot activation in ID cell formation and of KCa
channels in the generation of HD cells. These data are
further supported by the marked di€erence in the
contribution of F cells to each dehydrated fraction. In
the newly formed ID cells, the percentage of F cells is
only slightly lower than in the parent ND cells,
consistent with the HbF independence of KCl-cot
activity.3,19 In contrast, in the newly formed HD cells,
the percentage of F cells is much lower than in the
parent ND cells, supporting a role for sickling-induced
KCa-channel activation mostly in cells with low levels of
HbF.3 Although the conclusions derived are convergent
with those of others,18,19 the experimental conditions are
not physiological (pH 6.8 and long incubations).
Figure 2 shows a two-step pathway (sequential
activation of KCl-cot and KCa channels) for maximal
dehydration of T+F7 cells and a one-step pathway
(activation of KCl-cot) for intermediate dehydration of
T+F+ cells. This model also accounts for the
generation of ID and HD cells, by KCl-cot and KCa
channel activation, respectively and for their depen-
dence on HbF.
Figure 2 Mechanisms of SS RBC dehydration induced by acid
pH or deoxygenation. Two-step model for maximal dehydration
of T+F7 cells (or formation of HD cells from ND cells) and one-
step model for intermediate dehydration of T+F+ cells (or
formation of ID cells from ND cells). ND, normal density cells;
ID, intermediate density cells; HD, high density cells (the density
limits are indicated); T+F+ and T+F7, young RBC containing
transferrin receptors and HbF or no HbF, respectively; O-D
cycles, oxygenation-deoxygenation cycles; deoxy, deoxygenation.
The Hematology Journal
Initial activation of KCl-cot as the trigger of
dehydration does not support the previously proposed
model of reticulocyte dehydration,21 with initial
activation of KCa channels, slight acidi®cation and
secondary activation of KCl-cot. Such a mechanism is
not consistent with the activation of KCl-cot in the
absence of external Ca2+ during cyclic deoxygenation18
or during continuous deoxygenation in a fraction of
Mg2+-clamped light SS RBC.7 It is neither contradicted
nor proved by the ®nding that, upon deoxygenation,
rapid dehydration of young SS cells is strictly Ca2+-
dependent.3 However, it cannot be excluded that, in
some light cells (with low HbF content, large SIP-
induced Ca2+ in¯ux and high density of KCa channels),
activation of the channels could trigger activation of
the cotransport.
A role for cyclic vs continuous deoxygenation
In the absence of other stimuli such as low pH or cell
swelling, deoxygenation promotes dehydration of
unfractionated SS cells or T+ cells, through activation
of KCl-cot, but only during relatively slow O-D
cycling.18,22,23 The increase in intracellular Mg2+ free
concentration ([Mg2+]i) induced by deoxygenation, was
thought to be responsible for the inhibition of the
transporter under continuous deoxygenation.8 Indeed,
pharmacological loading of cells with Mg2+ inhibits the
activity of KCl-cot.24 Consistent with this hypothesis,
after clamping of [Mg2+]i with ionophore A23187,
continuous deoxygenation, at physiological pH, stimu-
lates KCl-cot in low-density SS RBC.7 Activation of
KCl-cot by deoxygenation might result from a depho-
sphorylation of the transporter or its regulators7,18 (see
below), but would be attenuated by the concomitant
rise in [Mg2+]i. Upon reoxygenation, KCl-cot could
express its activity, assuming that [Mg2+]i decreases in a
shorter time than that required for rephosphorylation.
Under fast O-D cycling (30 ± 40 s periods approaching
those in vivo), cells would not remain in the deoxy state
for a sucient time to allow dephosphorylation and
KCl-cot activation upon subsequent reoxygenation.23
However, there is good evidence that this pathway
contributes to SS RBC dehydration in vivo, as shown
by the bene®cial e€ect of oral administration of
magnesium on SS RBC hydration (see below). KCl-
cot might be activated in SS RBC subjected to `slower-
than-normal' O-D cycles due to adherence to post-
capillary venules.
The role of deoxygenation-induced increase in
[Mg2+]i on the inhibition of KCl-cot remains con-
troversial. Such an increase in SS RBC (from 0.4 to
0.7 mM)25 is too small to signi®cantly a€ect the activity
of KCl-cot, at least when activated by cell swelling or
acid pH.24,26 The activation of the KCl-cot, after
[Mg2+]i clamping,7 could result from the removal of
other inhibitory divalent cations by the ionophore
treatment. Mn2+ is a likely candidate, as it inhibits the
transporter activity with a much higher anity than
Mg2+.24 Total Mn2+ concentration in RBC is 3.4 mM.

Assuming that the bu€ering capacity of RBC for Mn 2+
is the same as that for Mg2+, the free concentration
would be about 1 mM, and sucient to promote an
inhibition of the transporter, especially under deox-
ygenated conditions.
In RBC of many species (®sh, horse and sheep) and
in normal human RBC, O2 exerts an overriding control
on the activity of the KCl-cot (see references in Gibson
et al.27). In the absence of a suciently high PO2, KCl-
cot is inactive and refractory to stimuli such as low pH
or cell swelling. The O2 dependence of KCl-cot is not
mediated via changes in [Mg2+]i. The presence of an O2
sensor has been speculated to explain the activation of
the transporter. In contrast, in SS RBC, KCl-cot
remains capable of responding to these stimuli, even at
low PO2.8,27 The di€erent O2 dependence of KCl-cot in
SS RBC may be related to the interaction of the
abnormal Hb with the transporter,28 to the associated
properties of HbS (auto-oxidation, polymerization) or
to secondary changes in the activity of kinases/
phosphatases (see below).
In contrast to KCl-cot, activation of KCa channels
does not require a period of reoxygenation. Indeed, a
similar percentage of dehydrated T+ cells (as estimated
from the density score) is generated through KCa-
channel activation, with either 4 h continuous or cyclic
deoxygenation at pH 7.4.18 Continuous deoxygenation
of ND cells, at pH 6.8, for 22 h promotes HD cell
formation, by KCa-channel activation, to a greater
extent than O-D cycling.20 Instant state measurement of
KCa-channel activation demonstrates that each deox-
ygenation pulse, either single or repeated, causes a
reversible, sustained SIP-induced Ca2+ in¯ux, channel
activation and dehydration of 10 ± 45% of SS
discocytes (1.095 ± 1.106 g/mL) to dense cells
(41.118 g/mL).9 However, continuous deoxygenation
causes only one early and limited density shift, whereas
frequent O-D cycling generates a progressive increase
in the fraction of dehydrated cells. This contradiction
with the above mentioned data,18 may arise from
di€erences in the experimental conditions or from the
lower HbF content of T+ cells, when compared with
that of SS discocytes (Figure 1A,B). T+ cells are
predicted to be more sensitive to sickling-induced, KCa-
channel-mediated dehydration, than the discocytes and
a quantitative di€erence in the percentage of dehy-
drated cells, formed by continuous or cyclic deox-
ygenation, to be less easily detectable.
Phosphorylation/dephosphorylation: a signal to
activate K+ transporters?
The major erythroid KCl-cot (KCC1) is a member of
the KCC family, including up to now four isoforms,
which di€er in their distribution in di€erent tissues and
in the presence of putative phosphorylation sites in
their N- and C-terminal cytoplasmic domains. The
human erythroid KCC1 contains several consensus
phosphorylation sites for casein kinase 2 and protein
kinase (PK) C, but has no protein tyrosine kinase
Hypothesis: sickle cell dehydration
P Merciris and F Giraud
203
(PTK) phosphorylation sites. Hence, KCl-cot activity
29
may be regulated by serine/threonine phosphorylation
on the transporter itself and by tyrosine phosphoryla-
tion on regulatory proteins.
Studies with speci®c inhibitors of serine-threonine
phosphatases PP1 and PP2A30 (calyculin A and
okadaic acid), or with inhibitor of serine-threonine
PK and PTK31 (staurosporine), have demonstrated that
dephosphorylation of the KCl-cot, or of a regulatory
protein, promotes activity, while phosphorylation
decreases activity. The targets and the identity of PP
and PK involved in the regulation of KCl-cot are not
yet clearly characterized and appear to depend on the
stimulus. As the activation by staurosporine, or
n-ethylmaleimide (NEM), is fully inhibited and
reversed by calyculin A, the prevailing model is that
staurosporine and NEM can inhibit a kinase that lies
upstream to the step catalysed by the calyculin-
sensitive PP.31 ± 34 PTK of Src family (Fgr and Hck)
negatively regulate the transporter in mouse RBC and
are likely to be among the staurosporine- and NEM-
sensitive kinases.34 Consequently, the activation of
PP2A (and KCl-cot) by NEM32 would result from
NEM-induced inhibition of Src PTK. Mg2+ depletion
suppresses the NEM-induced activation of PP2A and
of the transporter,32 suggesting that the phosphatase
activation depends not only on the inhibition of Src
PTK, but also on the activity of another kinase
inhibited by removal of Mg2+. This latter kinase
might be a PTK of another family (Syk), as tyrphostin
B46 (a PTK inhibitor) blocks the transporter stimula-
tion induced by staurosporine or NEM in sheep RBC.33
Therefore, both inhibitory (Src) and activating (Syk)
PTK may be involved in regulating the activity of
PP2A and of the transporter. Activation of PP2A and
of KCl-cot by this mechanism is induced by inhibition
of Src (NEM or staurosporine) but requires a basal
activity of Syk, because it is suppressed when Syk is
inhibited (Mg2+ depletion). Mg2+ depletion (alone),
which inhibits PP1, does not stimulate PP2A,32
presumably as it suppresses the activity of both Src
Figure 3 Hypothetical model of KCl-cot activation by staur-
osporine, NEM or Mg2+ depletion in human RBC and by
deoxygenation in SS RBC. A represents the inactive form of the
transporter and B the active form. PP2A, phosphoserine-
threonine phosphatase type 2A; PSK, protein serine-threonine
kinase; PTK, protein tyrosine kinase (Src or Syk family). KCl-cot
can be activated by inhibition of Src PTK or PSK, or by
activation of Syk PTK.
The Hematology Journal
 Hypothesis: sickle cell dehydration
P Merciris and F Giraud
204
and Syk and, hence, would activate the transporter by
inhibiting a serine/threonine protein kinase (PSK)
which phosphorylates the dephosphorylated KCl-
cot.32,33 A model of regulation of PP2A and KCl-cot
in human RBC, integrating the data mentioned above,
is presented in Figure 3. Dephosphorylation of the
transporter (from state A to B) by PP2A induces its
activation and the reverse reaction, catalysed by a
PSK, promotes inactivation.
The mechanism of KCl-cot activation in deoxyge-
nated SS RBC remains speculative, but could involve
protein phosphorylation, as deoxygenation induces
both an okadaic acid-sensitive dephosphorylation of
membrane proteins,35 presumably corresponding to the
activation of PP2A, and a stimulation of PTK.36 More
speci®cally, deoxygenation stimulates Syk, but has no
e€ect on the Src kinase Lyn (P Merciris, MD Hardy-
Dessources and F Giraud, unpublished data). PTK
inhibitors (tyrphostins) reduce SIP-induced K+ e‚ux in
SS RBC, even in the absence of external Ca2+,
suggesting the involvement of PTK on the activity of
KCl-cot, KCa channels or both.36 A possible role for
Syk in the activation of PP2A and KCl-cot in
deoxygenated SS RBC would be consistent with the
model in Figure 3.
The regulation of KCa channels in RBC may involve
both PKA and PKC. Prostaglandin E2 (PGE2) and
endothelin-1 (ET-1), whose binding to speci®c recep-
tors triggers respectively PKA and PKC stimulation,
activate KCa channels.37,38 PGE2 activation presumably
results from a direct PKA-mediated e€ect on the
channels (or its regulator), as this metabolite is unable
to stimulate Ca2+ in¯ux (B Lakatos and L Varecka,
personal communication). ET-1 activates the channels
by increasing the maximum velocity and the Ca2+
sensitivity.38 This activation is associated with PKC
stimulation, as it is blocked by a PKC inhibitor,
suggesting that PKC activation could result in a
stimulation of Ca2+ in¯ux. The plasma levels of
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Preventing sickle cell dehydration is of potential
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for communicating their data. This work was supported by
funds from the French National Centre for Scienti®c
Research (CNRS, UMR 8619), the University of Paris
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Hypothesis: sickle cell dehydration
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