The Tropical Journal of Health Sciences, Vol. 1 (1994), pp.

33 – 47

PATHOLOGICAL AND CHEMICAL EFFECTORS OF THE ERYTHROCYTE CALCIUM-

PUMPING PROTEIN: A REVIEW

ENITAN A. BABABUNMI1, OLUFUNSO O. OLORUNSOGO1 AND CLEMENT O. BEWAJI2

1. Department of Biochemistry, University of Ibadan, Ibadan, Nigeria

2. Department of Physiology and Biochemistry, Faculty of Health Sciences, University
of Ilorin, Ilorin, Nigeria.

ABSTRACT
The regulation of intracellular Ca2+ concentration has become the
focus of research in many areas of biology and medicine in recent
years. The intracellular Ca2+ concentration controls (directly or
indirectly) many important cellular processes such as muscle
contraction and relaxation, nervous excitation, exocrine and endocrine
secretions, as well as complicated processes such as cell proliferation
and fertilization.
The Ca2+-pumping adenosine triphosphatase (Ca2+-ATPase) of the
plasma membrane and intracellular organelles is the primary enzyme
responsible for maintaining the very low intracellular level of Ca2+. How
the activity of this enzyme is modulated, and its role in the regulation
of intracellular Ca2+ is the main focus of this review. Alterations in the
regulation of the enzyme, and hence intracellular Ca2+ level, may play
key roles in pathological processes such as hypertension, kwashiorkor
and sickle-cell anaemia.

INTRODUCTION different cells and located in the

plasma membrane, mitochondria,
The regulation of intracellular
calcium concentration has Table 1: Calmodulin-dependent cellular reactions
and processes
generated widespread interest in
recent years primarily as a result of
Enzymes/Processes References
the recognition of the role of calcium
as a second messenger which Phosphorymase kinase 64
(glycogenolysis)
controls many vital cellular
Phospholipase A2 65
processes (Table 1). In many cells,
2+ Myosin light chain kinase 66
the free cytosolic Ca concentration
is maintained at levels about four Erythrocyte Ca2+-ATPase 2, 6, 67

orders of mafnitude lower than the Adenylate cyclase 68

free Ca2+ concentration in the Phosphodiesterase 21, 69
extracellular fluids. This is made Cell motility 70
possible by the presence of an ATP- Muscle contraction 71, 72
2+ 2+
driven Ca pump and a Na+/Ca
2+ Exo- and endocytosis 73
exchange system as well as Ca -
binding proteins and other Cell division and proliferation 74

components which are present in Fertilization 75

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 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

endoplasmic or sarcoplasmic THE ROLE OF CALCIUM AS AN

reticulum and in the cytosol (1). INTRACELLULAR MESSENGER

In the erythrocyte, the Ca2+- It is now generally accepted that

pumping protein (Ca2+-ATPase) is Ca2+ is a very important, perhaps the
generally regarded as the only most important, second messenger in
enzyme system responsible for living cells. It has also been suggested
maintaining the intracellular that Ca2+ may play the role of a
concentration of Ca2+ at levels much primary messenger since it directly
lower than in the plasma (2-5). The interferes with the generation of
concentration of Ca2+ in erythrocytes signals at the level of plasma
is usually 0.1M or less. On the other membranes by regulating K+, Na+ and
hand, its concentration in the even Ca2+ currents (1).
surrounding plasma is about 1 – 2
mM. Thus, a 10,000-fold gradient In multicellular organisms, each cell
exists across the erythrocyte must coordinate the wide range of
membrane. Ca2+ penetrates across activities within its cytoplasm in
the erythrocyte membrane into the concert with those of neighbouring
cytoplasms by passive diffusion. The cells.
efflux, however, is mediated by an
active process which pumps Ca2+ out, The need for intercellular
using ATP as an energy source. It has communica-tion is met by a set of
been demonstrated that the Ca2+- messenger molecules and cell surface
ATPase activity of the erythrocyte receptors that transduce the chemical
membrane and the active transport of messages into a recognisable signal.
Ca2+ are two manifestations of the The signal either activates or inhibits a
same process (6,7) biochemical reaction that is controlled
by a rate-limiting step, which is usually
The maintenance of a low
governed by a cellular regulator – a
concentration of Ca2+ within the
molecule that controls one or more
erythrocyte is necessary to prevent
critical processes. Hormones, cyclic
the rapid loss of K+ through a Ca2+-
nucleotides and calcium are the three
sensitive K+-channel in the erythrocyte
most important regulators or
membrane (8,9). More importantly,
messengers in mammalian systems,
Ca2+ has to fulfil some messenger
and their activities are interwoven.
functions inside the cell, hence
efficient transport systems are
A consequence of the messenger
required to maintain the Ca2+-gradient 2+
across the membrane (10). It is role of Ca is the necessity for its
therefore not surprising that the Ca2+- precise regulation. The control of the
pumping protein has been intensively intracellular level of Ca2+ is also an
and extensively studied during the essential step in metabolic
past decade. These research efforts regulation. Intracellular Ca2+
were recently rewarded by the concentration can be regulated by
elucidation of the complete primary transporting the ion across the
structure of the plasma membrane plasma membrane or by
Ca2+ pump by Verma et al. (11). redistribution within subcellular

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 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

organelles. The cytosol also In most eukaryotic cells, the same

contains a large number of membrane system may transport
2+
nonmembraneous ligands which are Ca by more than one of these
able to bind Ca2+. Some of these mechanisms (3). An extensive
ligands are simple compounds such discussion on the role of Ca2+
as adenine nucleotides, inorganic channels and exchangers is beyond
phosphate and citrate. Others are the scope of this review.
2+
proteins which are able to bind Ca
with high specificity and affinity (1-
3). However, these ligands may not GENERAL PROPERTIES OF THE
2+
be effective in the modulation of the ERYTHCOYE MEMBRANE Ca -
2+ ATPase
cytosolic free Ca concentrations in
concert with physiological demands.
Although the Ca2+-ATPase is
The maintenance of intracellular presumambly present in the plasms
2+
Ca at a level much lower than in membrane of most animal cells, the
the extracellular medium must enzymehas been most extensively
depend on its extrusion through the studied in the mammalian
plasma membrane, to counteract erythrocytes for the following reasons:
the continuous passive influx, rather
than on sequestration within (i) availability in sufficient quantity;
intracellular organelles or binding to
other ligands. There are four basic (ii) easy and reproducible
mechanisms by which Ca2+ is preparation of both right-side-out
and inseide-out haemoglobin-free
transported across biological
2+ 2+ ghost membranes, and
membranes: Ca channels, Ca -
pumping ATPases, exchangers, and (iii) the lack of sub-cellular organelles
electrophoretic uniporters. (Table 2). facilitates the preparation of
relatively pure plasma
Table 2: Calcium transporting systems in membranes.
biological membranes.
The mammalian erythrocyte also
Transport Membrane types performs functions similar to those of
systems other cells; i.e. maintenance of
ATPase Plasma membrane
osmotic equilibrium, transport of
monovalent and divalent cations,
Sarcoplasmic reticulum
anions and substrates. In addition, it
Endoplasmic reticulum contains a spectrum of lipids and
Exchangers Plasma membrane (Na+/Ca2+) proteins which, in a general sense,
Inner mitochondrial membrane
are also found in other mammalian
plasma membranes such as in liver
(Na+/Ca2+; H+/Ca2+)
and heart.
Channels Plasma membranes
Due to the relative ease of
Electrophoretic Inner mitochondrial enzymatic assay, the erythrocyte
uniporters membranes
Ca2+-ATPase has been widely studied
in several laboratories. However, a
study of the enzyme in unfractionated

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 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

ghosts initially proved difficult as a ATPase by calmodulin which led to

result of the presence of another ATP- the eventual purification of the
splitting enzyme, the Mg2+-ATPase in enzyme (14).
the membrane. Purification of the
The properties of the Ca2+-ATPase
Ca2+-ATPase was also difficult
of the erythrocyte membrane
because of the lability of the enzyme,
resemble, in many respects, the
its low concentration in the
analogous enzyme in the
membrane, and the fact that its
sarcoplasmic reticulum of muscle cell
molecular weight and solubilisation
except that it is a bigger polypeptide
properties are similar to those of band
with a molecular weight of about
3 – one of the most abundant proteins
140,000 daltons and is regulated by
in the erythrocyte membrane (12,13).
calmodulin (14,15). Calmodulin has
However, the enzyme was
also been reported to stimulate the
subsequently purified by applying
ATP-dependent Ca2+ uptake into
affinity chromatographic techniques
inside-out vesicles prepared from
which involved the coupling of
human erythrocytes (23) The
calmodulin to a Sepharose 4B matrix
mechanism of activation of the pump
(14,15).
by calmodulin is illustrated in Fig. 1.
Erythrocyte ghost membranes are
The specific activities reported for
usually prepared by procedures which
the purified Ca2+-ATPase preparations
evolved from the classical method of
range from 9.0 – 186.0 moles/mg
Dodge et al. (16). This process
protein/hour, compared with the usual
involves the hypotonic lysis of the red
blood cells, followed by several values of 0.3 – 3.0 moles/mg
hypotonic washes to remove the protein/hour for whole membranes
haemoglobin and other cytoplasmic (15, 24-26). The purified preparation,
proteins. In 1973, Bond and Clough when reconstituted into artificial
(17) reported the presence of a liposomes, is activated by calmodulin
protein in red blood cell haemolysates about 7-fold, in analogy with the
which activated the Ca2+-ATPase. findings on the membrane-bound
This observation was later confirmed enzyme. Calmodulin shifts the purified
by other workers (18-20). The and membrane-bound ATPase from a
activator protein was studied in low Ca2+-affinity state (Km for Ca2+ 10-
anumber of laboratories and found to 14 M) to a high Ca2+ affinity state (Km
resemble the Ca2+-dependent protein for Ca2+ about 1 M) (24,27).
activator of cyclic nucleotide
phosphodiesterase which was first ACTIVATORS OF THE Ca2+-PUMP
discovered by Cheung (21). It
acquired a variety of names including Studies on the purified and
modulator protein and calcium- reconstituted enzyme have shown that
dependent regulator (CDR). Cheung calmodulin is not unique as an
et al. (22) coined the name calmodulin activator of the Ca2+-ATPase. The
for this protein and this name enzyme requires phospholipids for
eventually gained general acceptance. activity as shown by various
It was the discover of a direct delipidation experiments (12, 28, 29).
activation of the erythrocyte Ca2+- It has been found that the transition

36
 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

from the low to the high Ca2+-affinity INHIBITORS OF THE Ca2+-PUMP

state can be induced, in the absence
of calmodulin, by a variety of acidic The erythrocyte membrane Ca2+-
phospholipids (phosphatidylserine, transport is not inhibited by ouabain, a
diphosphatidylglyceron, specific inhibitor of the Na+, K+-
phosphatidylinositol and phosphatidic ATPase which mediates the transport
acid). The phospholipids increase the of sodium and potassium ions across
Vmax and decrease the Km (for Ca2+) the membrane (6). Oligomycin,
of the isolated enzyme to the same caffeine, azide and fluoride also have
extent as calmodulin. Unsaturated no effect on the Ca2+ transport
fatty acids (oleic and linoleic acids) process (35). However, lanthanum
also have the same effect as the shows some selectivity for inhibition of
acidic phospholipids but at lower the Ca2+ transport protein, the Ca2+-
concentrations.. Neutral phospholipids ATPase (36). The trivalent
(phosphatidylcholine, lanthanides, holmium and
phosphatidylethanolamine and praseodymium, inhibit Ca2+ transport
sphingomyelin) have no effect on the and the Ca2+-ATPase (37) but the
enzyme (27, 30, 31). required concentrations are
sufficiently high to inhibit the Na+, K+-
The purified enzyme, after ATPase. Various sulphydryl blocking
reconstitution in phosphatidylcholine reagents such as mersalyl (6), 4-
liposomes in the absence of chloromercuribenzoate and N-ethyl
calmodulin, can slao be activated by maleimide (38) have been shown to
controlled proteolysis with trypsin and inhibit Ca2+ transport and/or the Ca2+-
chymotrypsin (27, 30). The trypsinized ATPase, but the inhibition is not
enzyme has the same high Vmax and specific. The Mg2+-activated and the
high affinity for Ca2+ as the untreated Na+, K+-activated ATPases are also
enzyme in the presence of inhibited by comparable
calmodulin. It has been suggested concentrations of the reagents.
that the essential machinery of the red Vincenzi (39) showed that ethacrynic
cell Ca2+ pump is contained in a single acid is inhibitory at 10-3M.
polypeptide with a molecular weight of
about 140,000 daltons, but this is Watson et al. (40) investigated the
normally turned off until it encounters effects of ruthenium red on ATPase
the right Ca2+-calmodulin complex. activities of isolated red blood cell
Interaction with anionic ampiphilic membranes. Ruthenium red was
molecules, whether fatty acids or found to inhibit the Ca2+-ATPase. It
phospholipids, may somehow mimic was suggested that ruthenium red
the effect of calmodulin; proteolysis might be a specific inhibitor of Ca2+
seems to remove the control site, transport (41).
leaving the enzyme fully active (30-
34). Vanadate (VO43-) has also been
reported to inhibit several ATPases,
including the Na+, K+-ATPase, the
Ca2+-ATPase of erythrocyte ghosts
and, at higher concentrations, the
Ca2+-ATPase of the sarcoplasmic

37
 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

reticulum (42-44). This effect of been suggested that NAP-taurine

vanadate has been attributed to its might be a specific inhibitor of the
structural similarity to phosphate. Ca2+-ATPase (49). The properties of
Other chemical substances which the erythrocyte membrane Ca2+-
have been reported to inhibit the Ca2+- ATPase are summarized in Table 3.
ATPase in a non-specific manner
include R 24571, a potent inhibitor of MODULATION OF THE Ca2+-PUM
calmodulin-activated enzymes (45), BY DISEASE
quercetin, phloretin, vinblastine,
phenothiazine neuroleptics and (i) Sickle cell anaemia
suramin – a drug used in the early
stages of trypanosomiasis, filariasis Sickle cell anaemia is an inherited
and onchocerciasis (46). disease associated with the presence
in the red blood cell of sickle cell
It has also been reported that haemoglobin (HbS) one amino acid
inhibitors of the erythrocyte membrane residue of which is different from
band 3 (anion channel) protein such normal adult haemoglobin. The
as 4-acetamido-4-isothiocyano-2,2’- difference lies in the sixth amino acid
stilbene disulphonic acid (SITS), 4,4’- of the beta-chain where valine is
diisothiocyano-2,2’-stilbene substituted for glutamic acid of normal
disulphonic acid (DIDS) and N-(4- adult haemoglobin. In addition, the
azido-2-nitrophenyl)-2-aminoethyl Ca2+ regulation in sickle cells appears
sulphonic acid (NAP-taurine), inhibited to be disturbed.
the purified and reconstituted Ca2+-
ATPase of erythrocytes (47-49). It has Some investigators have reported
that the total Ca2+ content of sickle
Table 3: General properties of the erythrocyte cells is increased up to eight fold,
membrane Ca2+-ATPase.
compared to that of normal
erythrocytes (50,51). Treatment of
Relative molecular About 140,000
mass normal red cells with Ca2+ and the
specific Ca2+-ionophore A23187
Classification P-type ATPase, forming
phosphorylated
mimics known characteristics of
intermediates of the E1-E2 irreversibly sickled cells (52). This
type. suggests that the elevated Ca2+ level
Km (Ca2+) Low affinity, > 10M may play a role in the pathology of
sickle cell anaemia.
High affinity, < 1M
Activators Acidic phospholipids, Recent studies have shown that the
calmodulin, polyunsaturated
fatty acids, phosphorylation sickle cell membrane-bound Ca2+-
by protein kinases. ATPase has a Vmax which is
Inhibition Vanadate (Ki about 3M)
considerably lower than that of the
constants Lanthanum (Ki about 1M) normal enzyme (53). Gopinath and
Vincenzi (19) have also found that the
Stoichiometry Ca2+/ATP (1:1);
electroneutral exchange of Ca2+-ATPase of calmodulin-deficient
Ca2+ for H+. sickle cell membranes show a lower

38
 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

Fig. 1: The enzyme cycle of (A) Sarcoplasmic reticulum, and (B) plasma membrane Ca2+-ATPase.
E1 and E2 represent different conformational states of the enzyme.

39
 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

(Calmodulin)inactive + Ca2+  (Calmodulin*.Ca2+)active

(ATPase)less active + (Calmodulin*.Ca2+)active 

(ATPase*.Calmodulin*.Ca2+)active

Fig. 2: Mechanism of activation of the erythrocyte Ca2+-ATPase by Ca2+ and calmodulin. The
asterisk indicates a new conformation.

40
 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

Fig. 3: A chemiosmotic model for the mechanism of Ca2+ translocation across the erythrocyte
membrane, showing (A) a Fluid-Mosaic representation of Ca2+ fluxes across the
membrane and, (B) the balance of charges during the operation of the Ca2+ pump.
act = activator of the pump.

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 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

response to activation by calmodulin (i) Kwashiorkor

than the normal enzyme. This finding
has been confirmed by Dixon and Kwashiorkor is a disease of protein-
Winslow (54) who fould a lower energy malnutrition (PEM). It arises as
specific activity, and no calmodulin a result of quantitative and qualitative
stimulation, of the Ca2+ pump in sickle deficiency of dietary protein coupled
cell ghosts. Litosch and Lee (55) have with a grudgingly adequate energy
also studied the Ca2+-ATPase activity intake. The disease is characterized
of calmodulin-depleted sickle cell by oedema, a low body weight for
membranes. They showed that age, dermatosis, hair loss, mental
calmodulin increased the Ca2+- retardation, hepatomegaly and
ATPase activity of normal erythrocytes diarrhoea (58).
by about 200%, and the sickle cell
The activity of the Ca2+-pumping
Ca2+-ATPase by about 140%.
ATPase has been found to be at least
Bookchin and Lew (56) have 10% less in erythrocytes of
measured Ca2+-efflux from Ca2+- kwashiorkor patients than in normal
loaded normal and sickle cells and erythrocytes (59). The implication of
reported that in contrast to normal red this finding is that there will be a
cells, a substantial fraction of the raised intracellular Ca2+ level in
sickle cell population exhibited weak kwashiorkor as a result of the
Ca2+ pump activity. This observation defective Ca2+ pump.
was more pronounced in
(ii) Hypertension
deoxygenated sickle cells. They
postulated that sickling causes a
This is an important predisposing
progressive Ca2+ pump failure and a
“intermediate” to several other major
marked reduction in the Ca2+:Ca2+
diseases such as atherosclerosis.
exchange. They concluded that the
However, it is amenable to treatment.
increased intracellular Ca2+ and
If untreated,moderate hypertension
apparent Ca2+ pump failure in sickle
eventually leads to cardiac failure,
cells may be due to endocytic vesicles
with dilation of the left ventricle and
which trap Ca2+ in these cells. Bewaji
congestion of the pulmonary or
et al. (57) have also reported that the
systemic veins. Itcould also lead to
sickle cell membrabe-bound Ca2+-
nephrosclerosis.
ATPase can be activated by an
antisickling agent, 3,4-dihydro-2,2- It has been reported that the basal
dimethyl-2H-1-benzopyran-6-butyric activities of the erythrocyte Ca2+-
acid (DBA) in a Ca2+-dependent ATPase in hypertensive and
manner. They suggested that the normotensive individuals are the
antisickling effect of DBA may be same. However, calmodulin does not
connected with the mobilization of activate the ATPase of hypertensive
Ca2+ within red cells. This supports patients as much as it does that of
the assertion that the elevated normotensive individuals (60).
intracellular Ca2+ level plays a great
role in the pathology of sickle cell
anaemia.

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 The Tropical Journal of Health Sciences, Vol. 1 (1994), pp. 33 – 47

CONCLUDING REMARKS in membrane rigidity and eventual

destruction of the cell. The activity of
Bababunmi et al. (61) in a recent the Ca2+-ATPase in senescent red
review, have suggested that the cells and in pathological conditions
characterization of the plasma such as sickle cell anaemia,
membrane Ca2+ transporting ATPase hereditary spherocytosis and cystic
may be useful in understanding the fibrosis, therefore, needs to be further
pathobiology of diseases such as investigated. This would provide
malnutrition, parasitic diseases, information on the reasons for the
genetic disorders and cardiovascular operation of the Ca2+ pump below its
diseases. The function of this enzyme maximum capacity under
is to pump out Ca2+ from the cell physiological conditions.
thereby keeping the intracellular Ca2+
concentration very low.
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