Inhibition of SERCA Ca
2+
pumps by 2-aminoethoxydiphenyl borate
(2-APB)
2-APB reduces both Ca
2+
binding and phosphoryl transfer from ATP, by interfering
with the pathway leading to the Ca
2+
-binding sites
Jonathan G. Bilmen, Laura L. Wootton, Rita E. Godfrey, Oliver S. Smart and Francesco Michelangeli
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK
2-Aminoethoxydiphenyl Borate (2-APB) has been exten-
sively used recently as a membrane permeable modulator
of inositol-1,4,5-trisphosphate-sensitive Ca
2+
channels and
store-operated Ca
2+
entry. Here, we report that 2-APB is
also an inhibitor of sarco/endoplasmic reticulum Ca
2+
-
ATPase (SERCA) Ca
2+
pumps, and additionally increases
ion leakage across the phospholipid bilayer. Therefore, we
advise caution in the interpretation of results when used in
Ca
2+
signalling experiments. The inhibition of 2-APB

near or between transmembrane helices M3, M4, M5 and
M7. The binding of 2-APB to these sites could influence
the movement of the loop between M6 and M7 (L6-7), and
reduce access of Ca
2+
to their binding sites.
Keywords:2-APB;Ca
2+
-ATPase; Inhibition; SERCA.
Ca
2+
plays a very important role in a number of signalling
pathways, both within and between cells. The modulation
of its levels in the cytosol is crucial to the viability and
survival of the cell. Prolonged exposure to Ca
2+
can result
in apoptosis, whereas a lack of rise in cytosolic [Ca
2+
]may
lead to the failure of signal transduction [1]. Specific
pharmacological agents have been of great use as probes
to aid our understanding of Ca
2+
signalling processes [2–4].
One such agent, 2-aminoethoxydiphenylborate (2-APB),
has been reported to be a membrane permeable inhibitor of
the inositol-1,4,5-trisphosphate (InsP
3
)-sensitive Ca

3
,anIC
50
value of
220 l
M
was observed, while at 10 l
M
InsP
3
, the concentra-
tion of 2-APB required to half maximally inhibit Ca
2+
release is  1m
M
.
2-APB and xestospongin C (another cell permeant
InsP
3
receptor inhibitor) have been used to characterize
the mechanism of store-operated Ca
2+
entry, whereby
Ca
2+
influx from the extracellular matrix is triggered by
the emptying of Ca
2+
stores [7–10]. The concentrations of
2-APB used in these studies were in the range of

effects of 2-APB on the reduction of Ca
2+
efflux from
stores cannot also be discounted.
The SERCA family of pumps has been studied exten-
sively over the last 20 years and their mechanism of action
has been investigated by the use of inhibitors [13–16].
SERCA transports Ca
2+
ions across a lipid membrane
from the cell cytosol into distinct regions of the endoplas-
mic/sarcoplasmic reticulum. This transfer can be described
in terms of a scheme whereby the enzyme exists in two
conformational forms: a high affinity Ca
2+
binding (E1)
form, and a low affinity Ca
2+
binding (E2) form [17]. The
enzyme can cycle between these forms, transporting Ca
2+
ions, at the expense of ATP hydrolysis.
Correspondence to F. Michelangeli, School of Biosciences,
University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK.
Fax: + 44 121 414 5925, Tel.: + 44 0121 414 5398,
E-mail:
Abbreviations: 2-APB, aminoethoxydiphenyl borate; IC
50
,
concentration inducing half-maximal inhibition; EC

M4 and M6. More recently, Lee & East suggested an
alternative pathway, whereby M1 may form part of the
Ca
2+
channel leading to the binding sites [19]. Mutagenesis
studies have also implicated the M6–M7 loop (L6–7) and
regions of M3 as the Ca
2+
entry pathway/gateway [20,21].
Here, we present data to show that 2-APB can inhibit the
SERCA Ca
2+
pumps by reducing both the affinity of Ca
2+
binding and phosphoryl transfer and postulate that the drug
binds to and interferes with the Ca
2+
entry pathway of the
Ca
2+
-ATPase.
MATERIALS AND METHODS
2-Aminoethoxydiphenylborate (diphenylboric acid
2-amino-ethyl ester or 2-APB) was purchased from Sigma.
[c-
32
P]ATP was obtained from Amersham. Vector plasmids
containing both wild-type and mutant cDNA for the rabbit
skeletal muscle SR Ca
2+

COS-7 cells that were not transfected, and it was found that
Ca
2+
-dependent ATPase activity in the microsomal extracts
was £ 10% of those microsomes harvested from transfected
cells, indicating at least a 10-fold higher expression of
transfected Ca
2+
-ATPase to endogenous enzyme.
Ca
2+
-ATPase activity
The Ca
2+
-dependent ATPase activity in a number of
experiments involving microsomes or skeletal muscle SR
were performed using the phosphate liberation assay as
described by Longland et al. [25]. Briefly, microsomal
extracts (50 lg of cerebellar protein or 1 lgofSRprotein)
were re-suspended in 1 mL of buffer containing 45 m
M
Hepes/KOH (pH 7.0), 6 m
M
MgCl
2
,2m
M
NaN
3
,0.25

measurements involving microsomal extracts of transfected
COS-7 cells, the same procedure was followed, but was
miniaturized by 10-fold due to the low amount of enzyme
present (microsomal protein concentration of 40 lgÆmL
)1
was used for the assays).
Additional experiments, where the effects of 2-APB on
the activity of the purified Ca
2+
-ATPase were investigated,
were carried out using a coupled enzyme assay as previously
described [22]. Typically, 15 lgofATPaseproteinwas
added to a buffer containing 40 m
M
Hepes/KOH, 5 m
M
MgSO
4
,0.42m
M
phosphoenolpyruvate, 0.15 m
M
NADH,
7.5 U pyruvate kinase, 18 U lactate dehydrogenase,
1.01 m
M
EGTA and 2.1 m
M
ATP at pH 7.2. In experi-
ments performed at pH 6.0, in 50 m

Effects of 2-APB on FITC-labelled Ca
2+
-ATPase
Purified ATPase was labelled with fluorescein 5¢-isothio-
cyanate (FITC), according to the method described by
Michelangeli et al. [15], to monitor the E2 fi E1 transi-
tion. The purified ATPase was added in equal volume to
the starting buffer (1 m
M
KCl, 0.25
M
sucrose and 50 m
M
potassium phosphate pH 8.0). FITC in dimethylforma-
mide was then added at a molar ratio of FITC/ATPase,
0.5 : 1. The reaction was incubated for 1 h at 25 °C and
stopped by the addition of 0.25 mL of stopping buffer
(0.2
M
sucrose, 50 m
M
Tris/HCl pH 7.0), which was left
to incubate for 30 min at 30 °C prior to being placed on
ice until required. Fluorescence measurements of FITC-
ATPase was in a buffer containing 50 m
M
Tris, 50 m
M
maleate, 5 m
M

1m
M
CaCl
2
in a total
volume of 1 mL. ATP stocks (0.5 and 5 m
M
)weremade
in the buffer to cover a range of ATP concentrations
(specific activity 100 and 10 CiÆmol
)1
, respectively). The
reaction was initiated by addition of the appropriate
amounts of [c-
32
P]ATP and inactivated 15 s later by the
addition of 250 lL ice-cold 40% (w/v) trichloroacetic
acid. The samples were placed on ice for 30 min
subsequent to the addition of BSA (final conc.
0.5 mgÆmL
)1
). Purified ATPase was separated from the
solution by filtration through Whatman GF/C filters. The
filters were washed with 12% (w/v) trichloroacetic acid/
0.2
M
H
3
PO
4

2+
-induced
conformational changes
The conformational change induced by addition of Ca
2+
to
the ATPase was observed by monitoring the change in the
intrinsic tryptophan fluorescence [13]. Purified ATPase was
used at a concentration of 0.5 l
M
to a buffer containing
20 m
M
Hepes/Tris, 100 m
M
MgSO
4
, 100 l
M
CaCl
2
(pH 7.0). In experiments performed at pH 6, the buffer
contained 50 m
M
Mes/KOH, 100 m
M
MgSO
4
,and1m
M

at 280 nm and measuring the emission above 320 nm using
a cut off filter. The Ca
2+
-binding conformation was
measured at pH 7.2 in 20 m
M
Hepes/Tris, 100 m
M
KCl,
5m
M
MgSO
4
,50l
M
EGTA plus 1 m
M
Ca
2+
(final conc.)
from syringe B. The Ca
2+
dissociation conformation was
measured at pH 7.2 in 20 m
M
Hepes/Tris, 100 m
M
KCl,
5m
M

,500l
M
[
3
H]glucose (0.2 CiÆmol
)1
)and
100 l
M
45
CaCl
2
(3 CiÆmol
)1
). EGTA was then added to
vary the free Ca
2+
concentration. Samples were then
rapidly filtered through Millipore HAWP filters (0.45 lm).
Filters were then left to dry, after which 8 mL of
scintillant was added. The filters were then counted for
both
3
Hand
45
Ca
2+
. The amount of [
3
H]glucose trapped

inspection of the crystal structure for Ca
2+
-ATPase
(1eul.pdb) [18]. After assessing a number of sites for
possible binding of 2-APB, two potential binding pockets
of suitable size and shape were identified. To check that
the drug could be reasonably accommodated in the
identified pockets an energy minimization routine was
performed. After hydrogen atoms were added to the
protein its coordinates were kept fixed. The tripos force
field was used to represent the drug but the boron atom
and phenyl rings were held rigid at the ab initio optimized
geometry. Partial charges for the drug were obtained from
the Gaussian calculation and derived from
AMBER
4.1 for
the protein [28]. Solvation effects were represented by a
distance dependent dielectric constant. The energy mini-
mization procedure provides a useful check that a pocket
is large enough to accommodate the drug. Pictures of
bound drug were produced using the
VMD
and
RASTER
3
D
software packages [29].
3680 J. G. Bilmen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
RESULTS
Inhibition of Ca

to measure the rate of quenching
of calcein-loaded liposomes were performed in order to
assess whether 2-APB affected ion leakage across the lipid
bilayer. It was found that there was a substantial increase in
membrane permeability rate to Co
2+
ions in the presence of
2-APB (i.e. 500 l
M
2-APB increased the leak rate of the
liposomes by threefold). A sample trace from these exper-
iments can be seen in Fig. 2.
Inhibition of purified Ca
2+
ATPase
Figure 3 shows the inhibition of purified Ca
2+
ATPase at
both pH 7.2 (Fig. 3A) and pH 6.0 (Fig. 3A, inset) in the
presence of 2-APB using the coupled enzyme assay. The
IC
50
of 2-APB at pH 7.2 was 800 ± 100 l
M
, however, at
pH 6.0 the IC
50
was  70 l
M
. This represents a 12-fold

for the inhibitory phase of 0.35 ± 0.06 m
M
.
However, in the presence of 2-APB (800 l
M
), the V
max
was reduced to 5.9 ± 0.2 UÆmg
)1
, and stimulatory and
inhibitory K
m
values increased to 0.90 ± 0.03 and
0.77 ± 0.33 m
M
, respectively. These results therefore sug-
gest that 2-APB may affect Ca
2+
binding.
Figure 3C shows the inhibition of the purified ATPase by
2-APB at varying concentrations of ATP. As described
previously, the data can be fitted to a bi-Michaelis–Menten
equation [30,31].The high affinity Ôcatalytic siteÕ is where ATP
binds and phosphorylates the ATPase, while the low affinity
Ôregulatory siteÕ is involved in stimulating the rate at which the
ATPase cycles [31]. The data was fitted to curves with the
following kinetic parameters: In the absence of 2-APB,
the catalytic K
m
was 9.4 ± 1.6 l

m
was 1.3 ± 0.5 m
M
with a corresponding V
max
of 8.1±1.0UÆmg
)1
.Inthe
presence of 2-APB (800 l
M
), the data could be fitted
assuming, the K
m
for both catalytic and regulatory sites
were unchanged (i.e. 9.4 ± 2.8 and 1.3 ± 0.5 m
M
,respec-
tively). The V
max
values, however, were reduced. The
catalytic and regulatory V
max
values were 2.5 ± 0.1 and
5.4±0.4UÆmg
)1
, respectively. Therefore 2-APB appeared
to have no effect on the apparent K
m
for ATP, which suggests
that 2-APB is unlikely to be affecting ATP binding to the

2+
binding
To assess whether 2-APB has an effect on the conformational
changes associated with Ca
2+
binding to the ATPase,
tryptophan fluorescence was monitored in the absence and
presence of 2-APB at varying free Ca
2+
concentrations. The
change in tryptophan fluorescence induced by Ca
2+
has been
attributed to a change in E1 conformational states during the
process of Ca
2+
binding [32]. Figure 4A,B illustrates
the change in tryptophan fluorescence induced by Ca
2+
in
the presence and absence of 2-APB both at pH 6.0 and
pH 7.2. In all results, the DF
max
values did not significantly
change (i.e 9.7–10.1% DF
max
at both pH values). There was,
however, a decrease in the EC
50
values. At pH 7.2, in the

Measuring
45
Ca
2+
binding to the ATPase
To deduce whether 2-APB was directly affecting Ca
2+
binding,
45
Ca
2+
binding experiments were also performed
on the purified ATPase (Fig. 5). The binding curves fitted to
the data in Fig. 5 give similar B
max
values in the absence and
Fig. 3. Effects of 2-APB on the purified skeletal muscle Ca
2+
ATPase activity as a function of free [Ca
2+
], [ATP] and [Mg
2+
]. Activities of the Ca
2+
ATPase were measured at 37 °C, using the coupled enzyme assay, at either pH 7.2 (A) or pH 6.0 (inset). The activity of purified Ca
2+
ATPase was
also measured as a function of free [Ca
2+
](B);[ATP](C)and[Mg

2+
binding to the ATPase was also
altered by 2-APB. The Hill coefficient changed from
1.6 ± 0.2 in the absence of 2-APB to 0.9 ± 0.1 in the
presence of 3 m
M
2-APB. These results demonstrate that
2-APB inhibits Ca
2+
binding to the Ca
2+
ATPase in a
competitive manner, making it noncooperative in the
process.
Kinetics of conformational changes associated
with Ca
2+
binding and dissociation to the Ca
2+
-ATPase
The rate constants for the conformational changes associ-
ated with either Ca
2+
binding or Ca
2+
dissociation to the
ATPase were measured in the absence and presence 2-APB
(3 m
M
) at pH 7.2, by monitoring the changes in tryptophan

To determine whether 2-APB affects the E2 fi E1 transi-
tion of the ATPase, the fluorescence change induced by
Ca
2+
on FITC-labelled Ca
2+
ATPase was measured at
pH 6. Due to the effects of 2-APB on Ca
2+
binding, 1 m
M
Ca
2+
was added to ensure the complete transition from the
E2andE1step.AscanbeseeninFig.7,2-APBcauseda
decrease in the Ca
2+
-dependent FITC-ATPase fluorescence
change. In addition, the increase in fluorescence in going
from E1 to E2, due to the addition of 400 l
M
orthovana-
date, was also measured. The fluorescence increase associ-
ated with the addition of orthovanadate changed in the
presence of 3 m
M
2-APB, from 7.8 ± 0.2 to 10.4 ± 0.4%.
Taken together these experiments suggest that 2-APB
prefers to bind the ATPase in an E1 conformational state.
However, as these experiments were undertaken at pH 6

ptophan fluorescence were measured in the absence (j) and presence
of 300 l
M
2-APB (d)or3m
M
2-APB (s). Each data point represents
the mean ± SD of three or four determinations.
Fig. 5. Effects of 2-APB on
45
Ca
2+
binding to the purified ATPase.
Binding of
45
Ca
2+
to purified ATPase was measured as a function of
free Ca
2+
,intheabsence(j) and presence (s)of3m
M
2-APB, at
25 °C, pH 7.2. Each data point is the mean ± SD of three to five
determinations.
Ó FEBS 2002 2-APB inhibition of SERCA (Eur. J. Biochem. 269) 3683
ATP binding and phosphorylation of the Ca
2+
-ATPase
Figure 8A shows the effects of 3 m
M

and the results presented in Fig. 8B. As can be seen, little or
no change in TNP-ADP binding was observed in the
absence or presence of 3 m
M
2-APB (i.e. apparent
K
d
¼ 3.5 l
M
in both cases). These results therefore indicate
that this drug is unlikely to have an effect on nucleotide
binding but does reduce the phosphoryl transfer step of the
enzyme.
Effects of 2-APB on mutant Ca
2+
-ATPase activity
Upon initial analysis involving docking of 2-APB to the
Ca
2+
-ATPase (see Materials and methods) certain residues
were identified to putatively play a role in binding 2-APB to
the enzyme. Several of these residues had been previously
mutated [24] and these mutant Ca
2+
-ATPases were there-
fore used to test whether these residues played a part in the
binding of 2-APB to the enzyme. These mutant SERCA
pumps were expressed and harvested from COS-7 cells in
the form of microsomal extracts. The Ca
2+

2+
dissociation in the absence or presence
of 2-APB. Each data curve is the result of the average of at least 10
individual traces. The solid lines represent the best fits assuming a
mono-exponential process with the rate constants given in Table 1.
Table 1. Results of curve fitting to the kinetic data performed on Ca
2+
ion binding and dissociation. These values are presented as means ± SEM.
Data presented is a result of an average of 10–12 individual experiments.
Experiment k
obs
(s
)1
) Amplitude Goodness of fit (R
2
)
Ca
2+
binding 7.08 ± 0.11 8.98 ± 0.03 0.95
Ca
2+
binding + 2-APB 0.89 ± 0.09 10.28 ± 0.74 0.90
Ca
2+
dissociation 3.37 ± 0.07 )9.28 ± 0.07 0.98
Ca
2+
dissociation + 2-APB 12.98 ± 0.36 )10.56 ± 0.20 0.94
Fig. 7. Effects of 2-APB on the E2 to E1 conformational step. The
fluorescence change of FITC-labelled ATPase, induced by 1 m

trations above 200 l
M
. As mentioned previously, we have
shown that InsP
3
-induced Ca
2+
releasefromtype1InsP
3
receptors is also affected at similar concentrations [6].
Furthermore, research into store-operated Ca
2+
entry has
shown 2-APB to be an effective inhibitor at concentrations
of 10–100 l
M
[7–10]. We therefore advise caution when
interpreting results obtained with 2-APB, when it is used at
concentrations above 200 l
M
,onCa
2+
signalling processes.
As described in this study, 2-APB reduces the affinity for
Ca
2+
binding to the ATPase in a competitive manner and
inhibits phosphoryl transfer without affecting nucleotide
binding. Furthermore, the inhibition of ATPase activity by
2-APB is pH-sensitive with a low pH favouring increased

the enzyme, thereby increasing the effectiveness of 2-APB as
an inhibitor of ATPase activity.
Toyoshima et al. have identified the amino acids that
contribute towards the formation of the two high-affinity
Ca
2+
-binding sites (site I: T799, E771, N768, E908, D800;
site II: N796, A305, V304, E309, I307) and proposed the
Ca
2+
entry pathway/gateway to be formed by interactions
between transmembrane helices M2, M4 and M6 [18].
There is also evidence to suggest that the movement of the
transmembrane loop between M6 and M7 (L6–7) may be
Fig. 8. Effects of 2-APB on ATP-dependent phosphorylation and
nucleotide binding to the Ca
2+
ATPase. (A) shows the effects of
phosphorylation of the SR Ca
2+
ATPase at varying concentrations of
[c-
32
P]ATP, in the absence (j) and presence (s)of3m
M
2-APB. (B)
shows the spectroscopic change attributed to TNP-ADP binding to the
ATPase in the absence (j) and presence (s)of3m
M
2-APB, mea-

2+
-dependent ATPase activity, with some
mutants also inhibiting Ca
2+
binding [20]. In addition, these
experiments also showed that for some mutants there was a
decrease in the phosphoenzyme (E–P) intermediate, and
that this was not due to an effect on the dephosphorylation
step. As 2-APB inhibits both Ca
2+
ion binding and
phosphorylation in a similar fashion, it may imply that this
compound could be binding in a region near to the
L6–7 loop.
Results obtained from the mutant Ca
2+
-ATPase activity
studies identified Tyr837 as a critical residue for 2-APB
dependent inhibition of enzyme activity. Molecular model-
ing studies were undertaken to identify possibly 2-APB-
binding sites within the structure of the Ca
2+
-ATPase using
procedures and assumptions as described in Materials and
methods. Analysis of the interactions of 2-APB with the
structure of the ATPase identified two potential sites.
Figure 10 shows these binding sites in detail and highlights
the amino acids Tyr837, Phe834, Phe256 and Asn768.
One potential site is located between the top of trans-
membrane helix M7 and the middle of M3, with amino

binding and ATP-dependent phosphorylation of the
ATPase.
The fact that 2-APB decreases Ca
2+
bindingbyboth
reducing the rate constant for binding as well as increasing
therateconstantforCa
2+
dissociation, could be explained
by 2-APB binding to either one or both of these sites.
In summary, 2-APB is an inhibitor of the Ca
2+
-ATPase
that reduces its affinity for Ca
2+
and inhibits phospho-
enzyme formation, without affecting ATP binding. Fur-
thermore, from its mechanism of inhibition and from
molecular modeling studies we suggest that it may bind near
or between transmembrane helices M3, M4, M5 and M7
and propose that it influences the pathway leading to the to
the Ca
2+
-binding sites.
ACKNOWLEDGEMENTS
We would like to thank Dr J. Malcolm East and Prof C. David
O’Connor from the University of Southampton, UK for the SERCA
plasmids used in this study. We also thank the BBSRC for a PhD
studentship to J. G. B., the BHF for a PhD studentship to L. L. W.,
the MRC for the bioinformatics grant (64600017) and Dr Shahidul

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