Báo cáo Y học: Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB) 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites ppt

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

on the SERCA Ca
2+
pumpsisisoform-dependent,with
SERCA 2B being more sensitive than SERCA 1A (IC
50
values for inhibition being 325 and 725 l
M
, respectively,
measured at pH 7.2). The Ca
2+
-ATPase is also more
potently inhibited at lower pH (IC
50
¼ 70 l
M
for
SERCA 1A at pH 6). 2-APB decreases the affinity for
Ca
2+
binding to the ATPase by more than 20-fold, and
also inhibits phosphoryl transfer from ATP (by 35%),
without inhibiting nucleotide binding. Activity studies
performed using mutant Ca
2+
-ATPases show that Tyr837
is critical for the inhibition of activity by 2-APB. Molecular
modeling studies of 2-APB binding to the Ca
2+
ATPase
identified two potential binding sites close to this residue,

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

2+
channelwithanIC
50
value of 42 l
M
(in the presence of
100 n
M
InsP
3
) [5]. However, the effectiveness of 2-APB as a
modulator of the InsP
3
receptor (InsP
3
R) has recently been
questioned. We have recently shown that 2-APB is a lower
affinity inhibitor of the type 1 InsP
3
R than was originally
reported [6]. Our results show that the potency of 2-APB to
inhibit InsP
3
-induced Ca
2+
release is dependent upon InsP
3
concentration used. At 0.25 l
M
InsP

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

10–100 l
M
. One recent study has suggested that 2-APB
inhibits store-operated Ca
2+
entry into hepatocytes by
direct interaction with the store-operated Ca
2+
influx
channel, rather than by indirect effects on the InsP
3
receptor [11].
Missiaen et al. also recently reported that 2-APB inhibits
ATP-dependent Ca
2+
uptake in permeabilized A7r5 cells,
with an IC
50
of  90 l
M
[12]. Although Ca
2+
uptake in
intracellular Ca
2+
stores cells is primarily through the
actions of a group of transport proteins known as the sarco/
endoplasmic reticulum Ca
2+
-ATPases (SERCA), the

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

50
,
concentration inducing half-maximal stimulation; E–P
max
, maximal
level of phosphoenzyme formation; SERCA, sarco/endoplasmic
reticulum Ca
2+
-ATPase.
(Received 7 March 2002, revised 28 May 2002, accepted 18 June 2002)
Eur. J. Biochem. 269, 3678–3687 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03060.x
Toyoshima and coworkers have recently resolved the
crystal structure of the Ca
2+
-ATPase (SERCA 1A) to
2.6 A
˚
[18]. This resolution was of the ATPase in an E1 form,
with Ca
2+
bound. However, it was noted that there was no
obvious pathway through which Ca
2+
cantopass,inorder
to gain access to the Ca
2+
-binding sites embedded within
the transmembrane region of the protein. Toyoshima et al.
speculated that there could be a possible entry pathway
formed by amino acids on transmembrane domains M2,

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+

ATPase (SERCA 1) were received
as a gift from J. M. East and C. D. O’Connor (both from
the University of Southampton, UK). All other reagents
were of analytical grade. 2-APB was dissolved in dimeth-
ylsulfoxide to give a stock solution of 1
M
and the solvent
was never more than 0.3% (v/v) in the assays described.
Expression of the Ca
2+
-ATPase in COS-7 cells
COS-7 cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 0.11 gÆL
)1
sodium
pyruvate, pyridoxine (Gibco-BRL) and 10% fetal bovine
serum under 5% CO
2
/95% air at 37 °C. DNA transfection
was carried out using Transfast lipid transfection reagent
(Promega) following the instructions supplied.
Membrane and protein purification
SR and the purified Ca
2+
-ATPasewereextractedfrom
rabbit skeletal muscle, as described by Michelangeli &
Munkonge [22]. Cerebellar microsomes were prepared as
described by Sayers et al.[23].MicrosomesfromCOS-7
cells transfected with SERCA cDNA were prepared as
described previously [24]. Controls were performed with

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

M
sucrose, 12.5 lgÆmL
)1
A23187 ionophore, and EGTA with
CaCl
2
addedtogiveafree[Ca
2+
]of1l
M
. Assays were
preincubated at 37 °C for 10 mins prior to activation with
ATP (final conc. 6 m
M
). The reaction was stopped by
addition of 0.25 mL 6.5% (w/v) trichloroacetic acid. The
samples were put on ice for 10 min prior to centrifugation
for 10 min at 20 000 g. The supernatent (0.5 mL) was
added to 1.5 mL buffer containing 11.25% (v/v) acetic acid,
0.25% (w/v) copper sulphate, and 0.2
M
sodium acetate.
Ammonium molybdate [0.25 mL of 5% (w/v)] was then
added and mixed thoroughly. ELAN solution [0.25 mL,
consisting of 2% (w/v) p-methyl-aminophenol sulphate and
5% (w/v) sodium sulphite] was also added. The colour
intensity was measured after 10 min at 870 nm. Controls
were performed in the presence of dimethylsulfoxide, which
at maximal 2-APB concentrations was equal to 0.3% (v/v),
had no effect on the ATPase activity. For activity

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

M
Mes/KOH, 5 m
M
MgSO
4
,0.42m
M
phosphoenolpyruvate, 0.15 m
M
NADH,
22.5 U pyruvate kinase, 54 U lactate dehydrogenase and
1.01 m
M
EGTA were used. ATP at 2.1 m
M
was also
present. During ATP-dependent activity experiments, Ca
2+
was added to give the optimal activity (10 l
M
free Ca
2+
).
In additional experiments, where the Ca
2+
concentrations
were varied, the free Ca
2+
concentrations were calculated as
described in Gould et al. [26].

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

MgSO
4
and 100 m
M
KCl at either pH 6.0
or 7.0. Fluorescence was measured on a PerkinElmer
LS50B fluorescence spectrophotometer at 25 °C (excita-
tion 495 nm, emission 525 nm). EGTA, Ca
2+
,and
orthovanadate were then added to induce changes in
fluorescence intensity.
Ó FEBS 2002 2-APB inhibition of SERCA (Eur. J. Biochem. 269) 3679
Phosphorylation studies
Maximum levels of phosphorylation of the ATPase by
[c-
32
P]ATP was performed at 25 °C as described by
Michelangeli et al.[15].Briefly,SRwasdilutedto
75 lgÆmL
)1
in 20 m
M
Hepes/Tris (pH 7.2) containing
100 m
M
KCl, 5 m
M
MgSO
4,

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

, and left to dry, then placed in scintillant
and counted.
TNP-ADP binding to Ca
2+
-ATPase
The effects of 2-APB on the binding of a spectroscopic ATP
analogue, trinitrophenol adenosine diphosphate (TNP-
ADP), to SERCA was carried as described by Coll &
Murphy [27]. The purified ATPase was diluted to
0.8 mgÆmL
)1
in a buffer containing 20% (w/v) sucrose,
50 m
M
Mops/KOH (pH 7), 1 m
M
CaCl
2
.Thiswastitrated
with TNP-ADP in a Shimadzu UV-3000 dual wavelength
spectrophotometer and the absorbance was monitored at
422 and 390 nm, and the difference taken.
Membrane permeability studies
The effect of 2-APB on membrane permeability was
monitored by assessing the quenching of calcein dye trapped
in egg phosphatidylcholine liposomes by Co
2+
, as described
by Longland et al. [25].
Tryptophan fluorescence to follow Ca

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

CaCl
2
.Ca
2+
-associated fluorescence changes were calcu-
lated as a percentage of total fluorescence, by adding EGTA
and Ca
2+
to give known free Ca
2+
concentrations, based
on constants given previously [26]. Fluorescence was
measured on a PerkinElmer LS50B fluorescence spectro-
photometer at 25 °C (excitation 295 nm, emission 325 nm).
Measurement of the transient kinetics of the
conformational changes associated with Ca
2+
-binding
and dissociation
Rapid kinetic fluorescence measurements were performed
using a stopped-flow spectrofluorimeter (Applied photo-
physics, model SX17 MV) as described by Longland et al.
[13]. Briefly, the sample handling unit possesses two
syringes, A and B (drive ratio 10 : 1), which are driven by
a pneumatic ram. Tryptophan fluorescence was monitored
at 25 °C by exciting the 1 l
M
purified Ca
2+
ATPase sample

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

MgSO
4
,100l
M
Ca
2+
,plus2m
M
EGTA (final
conc.) from syringe B.
Measurement of
45
Ca
2+
-binding to the ATPase
45
Ca
2+
-binding to the ATPase was measured using the
dual labeling technique of Longland et al. [13]. ATPase
(0.1 mg) was incubated at 25 °C in 1 mL of buffer
containing 20 m
M
Hepes/Tris (pH 7.2), 100 m
M
KCl,
5m
M
MgSO
4

,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

on each filter was used to calculate the wetting volume
and was subtracted from the total Ca
2+
bound to the
filter, to give the specific amount of Ca
2+
bound to the
ATPase. A correction was also applied for nonspecific
binding of Ca
2+
to the lipid [13].
Modeling of the 2-APB-binding site on the Ca
2+
-ATPase
Molecular graphics and docking procedures were per-
formed principally with the
SYBYL V
6.5 package (Tripos
Inc). A Silicon Graphics Octane 2 workstation was used
for all graphics and calculations. The drug 2-APB was
initially sketched in
SYBYL
andthensubjectedtogeometry
optimization with
GAUSSIAN
98 (Gaussian Inc). A Har-
tree-Fock ab initio representation with a 3–21G basis set
was used. This provides a reasonable model for the drug
with estimates of partial atomic charges to allow docking
to be attempted. The docking procedure involved manual

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

2+
-ATPase activity and Ca
2+
uptake
Figure 1 shows the effects of 2-APB on Ca
2+
-dependent
ATPase activity, using the phosphate liberation method. As
can be seen from Fig. 1A,B, the Ca
2+
-ATPase activity
in cerebellar microsomes is inhibited with an IC
50
of
325 ± 19 l
M
. However, the IC
50
for SR Ca
2+
-ATPase
activity under the same conditions is 720 ± 45 l
M
.This
may be due to an isoform specific effect, as cerebellar
microsomes contain predominantly SERCA 2B, whereas
skeletal muscle SR contains SERCA 1 A.
2-APB affects membrane leakage
Experiments using Co
2+

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

change in IC
50
betweenpH6.0and7.2.
Experiments were then performed to see how 2-APB
affected ATPase activity at pH 7.2 as a function of Ca
2+
,
ATP and Mg
2+
. Figure 3B shows the effects of Ca
2+
on
ATPase activity, in the absence and presence of 800 l
M
2-APB. The data were fitted to the characteristic bell-shaped
curve of Ca
2+
-dependent ATPase activity. In the absence of
2-APB the ATPase had a V
max
of 11.4 ± 0.3 UÆmg
)1
,with
the K
m
for the stimulatory phase of 0.40 ± 0.03 l
M
,and
the K
m

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
,withaV
max
of
Fig. 1. Effects of 2-APB on Ca
2+
ATPase activity. The graphs repre-
sent Ca
2+
dependent ATPase activities, measured using the phosphate
liberation method in: (A) porcine cerebellar microsomes and (B) rabbit
skeletal muscle SR, measured at 37 °C,pH7.0.Eachdatapointisthe
mean ± SD of three determinations.
Fig. 2. 2-APB increases membrane permeability. The trace represents
experiments of Co
2+
quenching calcein trapped within liposomes. The
drop in fluorescence intensity over time represents quenching of the
fluorescent dye by Co
2+
ions (15 l
M
). Upon addition of 2-APB,
the rate of quenching is substantially increased and dependent upon
the concentration of 2-APB. The trace is representative of three or
more experiments.
Ó FEBS 2002 2-APB inhibition of SERCA (Eur. J. Biochem. 269) 3681
5.4±0.3UÆmg
)1
and the regulatory K

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

ATPase.
Figure 3D shows the inhibition of purified ATPase with
varying concentrations of Mg
2+
.Mg
2+
inhibits ATPase
activity at high concentrations with an IC
50
value of 8 m
M
,
which is not changed in the presence of 800 l
M
2-APB.
Again, the V
max
is decreased from 14.3to 7.2 UÆmg
)1
(taken
at the optimal [Mg
2+
]of2.5m
M
). Therefore, 2-APB is
unlikely to have any effect on Mg
2+
binding to the ATPase.
The effects of tryptophan fluorescence changes
associated with Ca

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

absence of 2-APB, the EC
50
was 0.6 ± 0.1 l
M
. Upon
addition of 3 m
M
2-APB, this value increased threefold to
1.7 ± 0.4 l
M
. At pH 6.0, the difference was even more
dramatic as the EC
50
value changed from 11.5 ± 0.1 l
M
in
the absence of 2-APB to 100 ± 11 and 350 ± 10 l
M
in the
presence of 300 l
M
and 3 m
M
2-APB, respectively. These
results therefore indicate that 2-APB affects the conforma-
tional changes associated with Ca
2+
binding to the Ca
2+
ATPase and that these effects are much greater at a lower pH.

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+
] (D), measured at pH 7.2, 37 °C in the absence (j) or presence (s) of 800 l
M
,
2-APB. Each data point is the mean ± SD of three to four determinations.
3682 J. G. Bilmen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
presence of 2-APB (20 ± 2 nmolÆmg
)1
in the absence of
2-APB and 21 ± 4 nmolÆmg
)1
the presence of 3 m
M
2-APB). The K
d
for Ca
2+
binding, although, were substan-
tially altered, as in the absence of 2-APB the K
d
was
0.4 ± 0.2 l
M
, while in the presence of 3 m
M
2-APB this
increased almost 20-fold to 6.9 ± 0.9 l
M
. In addition, The
cooperativity of Ca

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

fluorescence using stopped-flow spectrofluorimetry. In
Fig. 6A,B, the data were fitted to a mono-exponential
equation (rate constants given in Table 1) as this was the
simplest relationship which gave good fits to the data (i.e R
2
values ‡ 0.9). In Table 1, it can be seen that the rate
constant associated with Ca
2+
binding is decreased quite
dramatically in the presence of 2-APB (by nearly eightfold).
In addition, the rate constant for Ca
2+
dissociation in the
presence of 2-APB is also substantially increased by nearly
fourfold. As the K
d
for any binding process is related to
the ratio of k
off
/k
on
, a decrease in the rate constant for
Ca
2+
binding, and an increase in the rate constant for Ca
2+
dissociation will lead to a decreased affinity for Ca
2+
binding as shown earlier.
E2 fi E1 transition of the ATPase

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

where the IC
50
for the Ca
2+
-induced fluorescence changes
of the FITC-ATPase was calculated to be 1.5 m
M
, while the
IC
50
for ATPase inhibition at this pH is considerably less
(i.e. 70 l
M
), it is unlikely that the modulation of the E2 to
E1 step contributes greatly to the inhibition by 2-APB.
Fig. 4. Changes in tryptophan fluorescence of purified Ca
2+
ATPase, as
afunctionoffree[Ca
2+
] in the absence and presence of 2-APB. Purified
Ca
2+
ATPase (0.5 l
M
) was incubated in a buffer at either pH 7.2 (A)
or pH 6.0 (B) and the effects of different free [Ca
2+
] on the tryptophan
fluorescence intensities were performed at 25 °C. The change in try-

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

2-APB on the
phosphorylation of the Ca
2+
ATPase in SR. The EC
50
and maximum level of phosphoenzyme formation (E–P
max
)
in the absence of 2-APB was 11 ± 6 l
M
ATP and
1.5 ± 0.2 nmolÆmg
)1
ATPase, respectively. In the presence
of 3 m
M
2-APB, the EC
50
and E–P
max
were affected (i.e.
19 ± 6 l
M
ATP and 1.0 ± 0.1 nmolÆmg
)1
, respectively).
To investigate whether these effects could be due to
inhibition of the ATP binding step, or phosphoryl transfer
step, the binding of TNP-ADP, a nonhydrolyzable spec-
troscopic analogue of ATP, to the ATPase was measured

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+

-dependent
ATPase activity was measured in these microsomes in the
presence and absence of 800 l
M
2-APB (Fig. 9). In the wild-
type enzyme, 800 l
M
2-APB inhibited Ca
2+
-dependent
ATPase activity by  50% as expected. The activity of the
Phe834Ala SERCA mutant was reduced by about 60% in
the presence of 2-APB, though this difference was not
considered significant (P > 0.01). However, the activity of
the Tyr837Phe mutant SERCA pump was unaffected by the
presence of 800 l
M
2-APB (i.e. similar to controls). This
Fig. 6. Kinetics of the change in tryptophan fluorescence caused by the
binding/dissociation of Ca
2+
with purified ATPase. Experiments were
performed at 25 °C, pH 7.2. (A) shows the rate of change of the try-
ptophan fluorescence induced by Ca
2+
binding in the absence or
presence of 3 m
M
2-APB. (B) shows the rate of change of tryptophan
fluorescence induced by Ca

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

M
Ca
2+
, was measured as a function of 2-APB concentration, at 25 °C,
pH 6.0.
3684 J. G. Bilmen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
indicates a crucial role for the hydroxyl group of this
tyrosine residue in the binding of 2-APB to the enzyme.
DISCUSSION
2-APB has been used extensively recently to investigate the
effects of InsP
3
-induced Ca
2+
release and Ca
2+
influx in a
number of cell systems [11,12,33,34]. However, 2-APB also
inhibits SERCA pumps (especially SERCA 2B) and
increases membrane leakage that may cause artifactual
changes in intracellular [Ca
2+
] unrelated to its effects on
InsP
3
-sensitive Ca
2+
channels and store-operated Ca
2+
entry. These effects occur when 2-APB is used at concen-

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

inhibition. The structure of 2-APB is such that there is an
amino group on the end of an ethyl chain which would be
protonated, thereby having a positive charge (NH
3
+
)at
physiological pH. Depending upon the pKa of this amino
group, a change in pH may lead to different levels of
protonation, which may influence its ability to inhibit the
Ca
2+
-ATPase at different pH. However, as we have
experimentally determined the pKa of 2-APB to be 9.6, this
would mean that 2-APB would be virtually completely
protonated at both pH 7 or 6. A more plausible explanation
for the pH-sensitivity of 2-APB inhibition would therefore
be due to protonation of various amino-acid residues within
the ATPase at pH 6. Furthermore, as the rate limiting steps
within the enzymes cycle are different at pH 7 and pH 6
(Ca
2+
binding steps and dephosphorylation become rate
limiting at pH 6) [35]), the pH-dependence of inhibition can
be explained if 2-APB also specifically affects these steps.
However, it cannot be ruled out that the pKafor2-APB
when bound to the Ca
2+
-ATPase is significantly changed.
Therefore, it is possible that a decrease in pH may still lead
to protonation of an uncharged 2-APB molecule bound to

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-

sured by dual wavelength spectroscopy, at wavelengths 390 nm and
422 nm. The experiments were performed at 25 °C,pH7.2.Eachdata
point represents the mean ± SD of three to five determinations.
Fig. 9. Effects of 2-APB on Ca
2+
dependent ATPase activity of SR
Ca
2+
ATPase mutants expressed in COS-7 cells. Activities were
measured in the absence (black) and presence (white) of 800 l
M
2-APB, using the phosphate liberation assay, at 37 °C,pH7.2.Each
data point is the mean ± SD of three determinations. The activities of
the microsomes from these cells are typically between 40 and
80 nmolÆmin
)1
Æmg
)1
.
Ó FEBS 2002 2-APB inhibition of SERCA (Eur. J. Biochem. 269) 3685
responsible for the coupling of ion binding and phospho-
rylation [19,20,36,37]. In the crystal structure, it can be seen
that the L6–7 loop is in close proximity to the P2 helix of the
phosphorylation domain and the alignment of this loop has
been shown to change when the Ca
2+
ATPase is in a
vanadate-bound (E2) state. Furthermore, mutational
experiments involving the L6–7 loop have revealed a
decrease in Ca

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

acids on M5 and M4 also contributing to its binding (Site
A). As can be seen, the amino group of 2-APB bound in this
site is predicted to be close to Asn768. This residue is known
to constitute part of the Ca
2+
-binding site and plays a
crucial role in interacting with the Ca
2+
ions at both binding
sites [18]. This may therefore account for the effects of
2-APB on Ca
2+
binding. Also close to the drug in this site is
Phe256, which is known to be important for the effects of
another inhibitor, thapsigargin [38,39].
A second potential site has also been located from the
surface of the Ca
2+
-ATPase (Site B). As can be seen in
Fig. 9, the loss of the hydroxyl group from the Tyr837Phe
mutant enzyme resulted in a reduction of inhibition by
2-APB on ATPase activity. From the structure, it was
observed that the hydroxyl group was accessible from the
surface through a ÔchannelÕ. We could model 2-APB
interacting with this site, via a bridging water molecule
(Fig. 10). As can be seen, 2-APB binding to this site would
also be in close proximity to the L6–7 loop. Such an
interaction could explain the inhibitory effects of 2-APB on
Ca
2+

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

Islam for encouragement to undertake this study.
REFERENCES
1. Berridge, M.J., Bootman, M.D. & Lipp, P. (1998) Calcium – a life
and death signal. Nature 395, 645–648.
2. Waldron, R.T., Short, A.D. & Gill, D.L. (1997) Store-operated
Ca
2+
entry and coupling to Ca
2+
pool depletion in thapsigargin-
resistant cells. J. Biol. Chem 272, 6440–6447.
3. Brown, G.R., Sayers, L.G., Kirk, C.J., Michell, R.H. &
Michelangeli, F. (1992) The opening of the inositol 1,4,5-trispho-
sphate-sensitive Ca
2+
channel in rat cerebellum is inhibited by
caffeine. Biochem. J. 282 (2), 309–312.
4. Irving, A.J., Collingridge, G.L. & Schofield, J.G. (1992) Inter-
actions between Ca
2+
mobilizing mechanisms in cultured rat
cerebellar granule cells. J. Physiol. 456, 667–680.
5. Maruyama, T., Kanaji, T., Nakade, S., Kanno, T. & Mikoshiba,
K. (1997) 2APB, 2-aminoethoxydiphenyl borate, a membrane-
penetrable modulator of Ins (1,4,5), P3-induced Ca
2+
release.
J.Biochem.122, 498–505.
6. Bilmen, J.G. & Michelangeli, F. (2002) Inhibition of the type 1
inositol 1,4,5-trisphosphate receptor by 2-aminethoxydiphenyl-

borate (2-APB). Cell Signal,inpress.
7. Bakowski, D., Glitsch, M.D. & Parekh, A.B. (2001) An
examination of the secretion-like coupling model for the activation
of the Ca
2+
release-activated Ca
2+
current ICRAC in RBL-1
cells. J. Physiol. 532, 55–71.
8. Ma, H.T., Venkatachalam, K., Li, H.S., Montell, C., Kurosaki,
T., Patterson, R.L. & Gill, D.L. (2001) Assessment of the role of
the inositol 1,4,5-trisphosphate receptor in the activation of tran-
sient receptor potential channels and store-operated Ca
2+
entry
channels. J.Biol.Chem276, 18888–18896.
Fig. 10. Predicted sites of interaction of 2-APB with the Ca
2+
ATPase.
The figure shows two possible sites of interaction for 2-APB with the
ATPase, with the important residues highlighted and labelled.
3686 J. G. Bilmen et al. (Eur. J. Biochem. 269) Ó FEBS 2002
9. van Rossum, D.B., Patterson, R.L., Ma, H.T. & Gill, D.L. (2000)
Ca
2+
entry mediated by store depletion, S-nitrosylation, and
TRP3 channels. Comparison of coupling and function. J. Biol.
Chem 275, 28562–28568.
10. Ma, H.T., Patterson, R.L., van Rossum, D.B., Birnbaumer, L.,
Mikoshiba, K. & Gill, D.L. (2000) Requirement of the inositol

trisphosphate receptor for activation of store-operated Ca
2+
channels. Science 287, 1647–1651.
11. Gregory, R.B., Rychkov, G. & Barritt, G.J. (2001) Evidence that
2-aminoethyl diphenylborate is a novel inhibitor of store-operated
Ca
2+
channels in liver cells, and acts through a mechanism which
does not involve inositol trisphosphate receptors. Biochem. J. 354,
285–290.
12. Missiaen, L., Callewaert, G., De Smedt, H. & Parys, J.B. (2001)
2-Aminoethoxydiphenyl borate affects the inositol 1,4,5-trispho-
sphate receptor, the intracellular Ca
2+
pump and the non-specific
Ca
2+
leak from the non-mitochondrial Ca
2+
stores in permea-
bilized A7r5 cells. Cell Calcium 29, 111–116.
13. Longland, C.L., Mezna, M. & Michelangeli, F. (1999) The
mechanism of inhibition of the Ca
2+
-ATPase by mastoparan.
Mastoparan abolishes cooperative Ca
2+
binding. J.Biol.Chem
274, 14799–14805.
14. Hughes, G., Starling, A.P., Sharma, R.P., East, J.M. & Lee, A.G.

(1996) An investigation of the mechanism of inhibition of the
Ca
2+
-ATPase by phospholamban. Biochem. J. 318, 973–979.
15. Michelangeli, F., Orlowski, S., Champeil, P., East, J.M. & Lee,
A.G. (1990) Mechanism of inhibition of the (Ca
2+
-Mg
2+
)-
ATPase by nonylphenol. Biochemistry 29, 3091–3101.
16. Zhong, L. & Inesi, G. (1998) Role of the S3 stalk segment in the
thapsigargin concentration dependence of sarco-endoplasmic
reticulum Ca
2+
ATPase inhibition. J.Biol.Chem.273, 12994–
12998.
17. de Meis, L. (1981) The Sarcoplasmic Reticulum, 1st edn. John
Wiley and Sons, New York.
18. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000)
Crystal structure of the calcium pump of sarcoplasmic reticulum
at 2.6 A
˚
resolution. Nature 405, 647–655.
19. Lee, A.G. & East, J.M. (2001) What the structure of a calcium
pump tells us about its mechanism. Biochem. J. 356, 665–683.
20. Zhang, Z., Lewis, D., Sumbilla, C., Inesi, G. & Toyoshima, C.
(2001) The role of the M6–M7 loop (L67) in stabilization of the
phosphorylation and Ca
2+

binding domains of the sarcoplasmic
reticulum Ca
2+
-ATPase (SERCA). J. Biol. Chem. 276, 15232–
15239.
21. Andersen, J.P., Sorensen, T.L., Povlsen, K. & Vilsen, B. (2001)
Importance of transmembrane segment M3 of the sarcoplasmic
reticulum Ca
2+
-ATPase for control of the gateway to the Ca
2+
sites. J.Biol.Chem.276, 23312–23321.
22. Michelangeli, F. & Munkonge, F.M. (1991) Methods of recon-
stitution of the purified sarcoplasmic reticulum (Ca
2+
-Mg
2+
)-
ATPase using bile salt detergents to form membranes of defined
lipid to protein ratios or sealed vesicles. Anal. Biochem. 194,
231–236.
23. Sayers, L.G., Brown, G.R., Michell, R.H. & Michelangeli, F.
(1993) The effects of thimerosal on calcium uptake and inositol
1,4,5-trisphosphate-induced calcium release in cerebellar micro-
somes. Biochem. J. 289, 883–887.
24. Adams, P., East, J.M., Lee, A.G. & Connor, C.D. (1998) Muta-
tional analysis of trans-membrane helices M3, M4, M5 and M7 of
the fast-twitch Ca
2+
-ATPase. Biochem. J. 335, 131–138.

25. Longland, C.L., Mezna, M., Langel, U., Hallbrink, M., Soomets,
U., Wheatley, M., Michelangeli, F. & Howl, J. (1998) Biochemical
mechanisms of calcium mobilisation induced by mastoparan and
chimeric hormone-mastoparan constructs. Cell. Calcium 24, 27–
34.
26. Gould, G.W., East, J.M., Froud, R.J., McWhirter, J.M., Stefa-
nova, H.I. & Lee, A.G. (1986) A kinetic model for the Ca
2+
+
Mg
2+
-activated ATPase of sarcoplasmic reticulum. Biochem. J.
237, 217–227.
27. Coll, R.J. & Murphy, A.J. (1986) Affinity of nucleotides for the
active site of detergent-solubilized sarcoplasmic reticulum
CaATPase. Biochem. Biophys. Res. Commun 138, 652–658.
28. Cornell, W.D., Cieplak, P., Bayly, C.I., Gould, I.R., Merz, J.,
Ferguson, D.M., Spellmeyer, D.C., Caldwell, J.W. & Kollman,
P.A. (1995) A second generation force field for the simulation of
proteins, nucleic acids, and organic molecules. J.Am.Chem.Soc.
117, 5179–5197.
29. Humphrey, W.F., Dalke, A. & Schulten, K. (1996) VMD – Visual
Molecular Dynamics. J. Mol. Graph. 14, 33–38.
30. Dupont, Y., Pougeois, R., Ronjat, M. & Verjovsky, A. (1985)
Two distinct classes of nucleotide binding sites in sarcoplasmic
reticulum Ca-ATPase revealed by 2¢,3¢-O-(2,4,6-trinitrocyclo-
hexadienylidene)-ATP. J. Biol. Chem. 260, 7241–7249.
31. Coll, R.J. & Murphy, A.J. (1991) Kinetic evidence for two
nucleotide binding sites on the CaATPase of sarcoplasmic
reticulum. Biochemistry 30, 1456–1461.

32. Henderson, I.M., Starling, A.P., Wictome, M., East, J.M. & Lee,
A.G. (1994) Binding of Ca
2+
to the (Ca
2+
-Mg
2+
)-ATPase of
sarcoplasmic reticulum: kinetic studies. Biochem. J. 297 (3), 625–
636.
33. Wu,J.,Kamimura,N.,Takeo,T.,Suga,S.,Wakui,M.,Maruy-
ama, T. & Mikoshiba, K. (2000) 2-Aminoethoxydiphenyl borate
modulates kinetics of intracellular Ca
2+
signals mediated by
inositol 1,4,5-trisphosphate-sensitive Ca
2+
stores in single
pancreatic acinar cells of mouse. Mol. Pharmacol. 58, 1368–1374.
34. Ascher-Landsberg, J., Saunders, T., Elovitz, M. & Phillippe, M.
(1999) The effects of 2-aminoethoxydiphenyl borate, a novel
inositol 1,4,5-trisphosphate receptor modulator on myometrial
contractions. Biochem. Biophys. Res. Commun 264, 979–982.
35. Champeil, P. & Guillain, F. (1986) Rapid filtration study of
the phosphorylation-dependent dissociation of calcium from
transport sites of purified sarcoplasmic reticulum ATPase
and ATP modulation of the catalytic cycle. Biochemistry 25,
7623–7633.
36. Zhang, Z., Lewis, D., Strock, C., Inesi, G., Nakasako, M.,
Nomura, H. & Toyoshima, C. (2000) Detailed characterization of

the cooperative mechanism of Ca
2+
binding and catalytic acti-
vationintheCa
2+
transport (SERCA) ATPase. Biochemistry 39,
8758–8767.
37. Menguy, T., Corre, F., Bouneau, L., Deschamps, S., Moller, J.V.,
Champeil, P., le Maire, M. & Falson, P. (1998) The cytoplasmic
loop located between transmembrane segments 6 and 7 controls
activation by Ca
2+
of sarcoplasmic reticulum Ca
2+
-ATPase.
J.Biol.Chem273, 20134–20143.
38. YuM., Lin, J., Khadeer, M., Yeh, Y., Inesi, G. & Hussain, A.
(1999) Effects of various amino acid 256 mutations on sarco-
plasmic/endoplasmic reticulum Ca
2+
ATPase function and their
role in the cellular adaptive response to thapsigargin. Arch.
Biochem. Biophys. 362, 225–232.
39. YuM.,Zhang,L.,Rishi,A.K.,Khadeer,M.,Inesi,G.&Hussain,
A. (1998) Specific substitutions at amino acid 256 of the sarco-
plasmic/endoplasmic reticulum Ca
2+
transport ATPase mediate
resistance to thapsigargin in thapsigargin-resistant hamster cells.
J.Biol.Chem.273, 3542–3546.

Ó FEBS 2002 2-APB inhibition of SERCA (Eur. J. Biochem. 269) 3687