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+
Trang 1Inhibition 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
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 Ca2+channels and
store-operated Ca2+entry Here, we report that 2-APB is
also an inhibitor of sarco/endoplasmic reticulum Ca2+
-ATPase (SERCA) Ca2+pumps, and additionally increases
ion leakage across the phospholipid bilayer Therefore, we
advise caution in the interpretation of results when used in
Ca2+ signalling experiments The inhibition of 2-APB
on the SERCA Ca2+pumps is isoform-dependent, with
SERCA 2B being more sensitive than SERCA 1A (IC50
values for inhibition being 325 and 725 lM, respectively,
measured at pH 7.2) The Ca2+-ATPase is also more
potently inhibited at lower pH (IC50¼ 70 lM for
SERCA 1A at pH 6) 2-APB decreases the affinity for
Ca2+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 Ca2+-ATPases show that Tyr837
is critical for the inhibition of activity by 2-APB Molecular modeling studies of 2-APB binding to the Ca2+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 Ca2+to their binding sites
Keywords: 2-APB; C a2+-ATPase; Inhibition; SERCA
Ca2+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 Ca2+can result
in apoptosis, whereas a lack of rise in cytosolic [Ca2+] may
lead to the failure of signal transduction [1] Specific
pharmacological agents have been of great use as probes
to aid our understanding of Ca2+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 (InsP3)-sensitive Ca2+
channel with an IC50 value of 42 lM (in the presence of
100 nMInsP3) [5] However, the effectiveness of 2-APB as a
modulator of the InsP3receptor (InsP3R) has recently been
questioned We have recently shown that 2-APB is a lower
affinity inhibitor of the type 1 InsP3R than was originally
reported [6] Our results show that the potency of 2-APB to
inhibit InsP3-induced Ca2+release is dependent upon InsP3
concentration used At 0.25 lM InsP3, an IC50 value of
220 lMwas observed, while at 10 lMInsP3, the
concentra-tion of 2-APB required to half maximally inhibit Ca2+ release is 1 mM
2-APB and xestospongin C(another cell permeant InsP3 receptor inhibitor) have been used to characterize the mechanism of store-operated Ca2+ entry, whereby
Ca2+influx from the extracellular matrix is triggered by the emptying of Ca2+stores [7–10] The concentrations of 2-APB used in these studies were in the range of 10–100 lM One recent study has suggested that 2-APB inhibits store-operated Ca2+ entry into hepatocytes by direct interaction with the store-operated Ca2+ influx channel, rather than by indirect effects on the InsP3
receptor [11]
Missiaen et al also recently reported that 2-APB inhibits ATP-dependent Ca2+uptake in permeabilized A7r5 cells, with an IC50 of 90 lM [12] Although Ca2+ uptake in intracellular Ca2+ stores cells is primarily through the actions of a group of transport proteins known as the sarco/ endoplasmic reticulum Ca2+-ATPases (SERCA), the effects of 2-APB on the reduction of Ca2+ 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 Ca2+ 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 Ca2+ binding (E1) form, and a low affinity Ca2+binding (E2) form [17] The enzyme can cycle between these forms, transporting Ca2+ 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: F.Michelangeli@bham.ac.uk
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)
Trang 2Toyoshima and coworkers have recently resolved the
crystal structure of the Ca2+-ATPase (SERCA 1A) to
2.6 A˚ [18] This resolution was of the ATPase in an E1 form,
with Ca2+bound However, it was noted that there was no
obvious pathway through which Ca2+can to pass, in order
to gain access to the Ca2+-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
Ca2+channel leading to the binding sites [19] Mutagenesis
studies have also implicated the M6–M7 loop (L6–7) and
regions of M3 as the Ca2+entry pathway/gateway [20,21]
Here, we present data to show that 2-APB can inhibit the
SERCA Ca2+pumps by reducing both the affinity of Ca2+
binding and phosphoryl transfer and postulate that the drug
binds to and interferes with the Ca2+entry pathway of the
Ca2+-ATPase
M A T E R I A L S A N D M E T H O D S
2-Aminoethoxydiphenylborate (diphenylboric acid
2-amino-ethyl ester or 2-APB) was purchased from Sigma
[c-32P]ATP was obtained from Amersham Vector plasmids
containing both wild-type and mutant cDNA for the rabbit
skeletal muscle SR Ca2+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 1Mand the solvent
was never more than 0.3% (v/v) in the assays described
Expression of the Ca2+-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% CO2/95% air at 37C DNA transfection
was carried out using Transfast lipid transfection reagent
(Promega) following the instructions supplied
Membrane and protein purification
SR and the purified Ca2+-ATPase were extracted from
rabbit skeletal muscle, as described by Michelangeli &
Munkonge [22] Cerebellar microsomes were prepared as
described by Sayers et al [23] Microsomes from C OS-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
Ca2+-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 Ca2+-ATPase to endogenous enzyme
Ca2+-ATPase activity
The Ca2+-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 lg of SR protein)
were re-suspended in 1 mL of buffer containing 45 mM
Hepes/KOH (pH 7.0), 6 mMMgCl2, 2 mMNaN3, 0.25M
sucrose, 12.5 lgÆmL)1A23187 ionophore, and EGTA with CaCl2 added to give a free [C a2+] of 1 lM Assays were preincubated at 37Cfor 10 mins prior to activation with ATP (final conc 6 mM) 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 Ca2+-ATPase were investigated, were carried out using a coupled enzyme assay as previously described [22] Typically, 15 lg of ATPase protein was added to a buffer containing 40 mM Hepes/KOH, 5 mM
MgSO4, 0.42 mMphosphoenolpyruvate, 0.15 mMNADH, 7.5 U pyruvate kinase, 18 U lactate dehydrogenase, 1.01 mM EGTA and 2.1 mM ATP at pH 7.2 In experi-ments performed at pH 6.0, in 50 mM Mes/KOH, 5 mM
MgSO4, 0.42 mMphosphoenolpyruvate, 0.15 mMNADH, 22.5 U pyruvate kinase, 54 U lactate dehydrogenase and 1.01 mM EGTA were used ATP at 2.1 mM was also present During ATP-dependent activity experiments, Ca2+ was added to give the optimal activity (10 lMfree Ca2+)
In additional experiments, where the Ca2+concentrations were varied, the free Ca2+concentrations were calculated as described in Gould et al [26]
Effects of 2-APB on FITC-labelled Ca2+-ATPase Purified ATPase was labelled with fluorescein 5¢-isothio-cyanate (FITC), according to the method described by Michelangeli et al [15], to monitor the E2fi E1 transi-tion The purified ATPase was added in equal volume to the starting buffer (1 mMKCl, 0.25Msucrose and 50 mM
potassium phosphate pH 8.0) FITCin dimethylforma-mide was then added at a molar ratio of FITC/ATPase, 0.5 : 1 The reaction was incubated for 1 h at 25Cand stopped by the addition of 0.25 mL of stopping buffer (0.2 M sucrose, 50 mM Tris/HCl pH 7.0), which was left
to incubate for 30 min at 30Cprior to being placed on ice until required Fluorescence measurements of FITC-ATPase was in a buffer containing 50 mM Tris, 50 mM
maleate, 5 mMMgSO4and 100 mMKCl 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, Ca2+, and orthovanadate were then added to induce changes in fluorescence intensity
Trang 3Phosphorylation studies
Maximum levels of phosphorylation of the ATPase by
[c-32P]ATP was performed at 25Cas described by
Michelangeli et al [15] Briefly, SR was diluted to
75 lgÆmL)1 in 20 mM Hepes/Tris (pH 7.2) containing
100 mM KCl, 5 mM MgSO4, 1 mM CaCl2 in a total
volume of 1 mL ATP stocks (0.5 and 5 mM) were made
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-32P]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/Cfilters The
filters were washed with 12% (w/v) trichloroacetic acid/
0.2 M H3PO4, and left to dry, then placed in scintillant
and counted
TNP-ADP binding to Ca2+-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 mMMops/KOH (pH 7), 1 mMCaCl2 This was titrated
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 Co2+, as described
by Longland et al [25]
Tryptophan fluorescence to follow Ca2+-induced
conformational changes
The conformational change induced by addition of Ca2+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 lM to a buffer containing
20 mM Hepes/Tris, 100 mM MgSO4, 100 lM CaCl2
(pH 7.0) In experiments performed at pH 6, the buffer
contained 50 mMMes/KOH, 100 mM MgSO4, and 1 mM
CaCl2 C a2+-associated fluorescence changes were
calcu-lated as a percentage of total fluorescence, by adding EGTA
and Ca2+to give known free Ca2+concentrations, based
on constants given previously [26] Fluorescence was
measured on a PerkinElmer LS50B fluorescence
spectro-photometer at 25C(excitation 295 nm, emission 325 nm)
Measurement of the transient kinetics of the
conformational changes associated with Ca2+-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 25Cby exciting the 1 lMpurified Ca2+ATPase sample
at 280 nm and measuring the emission above 320 nm using
a cut off filter The Ca2+-binding conformation was measured at pH 7.2 in 20 mM Hepes/Tris, 100 mM KCl,
5 mMMgSO4, 50 lMEGTA plus 1 mMCa2+(final conc.) from syringe B The Ca2+dissociation conformation was measured at pH 7.2 in 20 mM Hepes/Tris, 100 mM KCl,
5 mM MgSO4, 100 lM Ca2+, plus 2 mM EGTA (final conc.) from syringe B
Measurement of45Ca2+-binding to the ATPase
45Ca2+-binding to the ATPase was measured using the dual labeling technique of Longland et al [13] ATPase (0.1 mg) was incubated at 25Cin 1 mL of buffer containing 20 mM Hepes/Tris (pH 7.2), 100 mM KCl,
5 mM MgSO4, 500 lM [3H]glucose (0.2 CiÆmol)1) and
100 lM 45CaCl2 (3 CiÆmol)1) EGTA was then added to vary the free Ca2+ 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 both3H and45Ca2+ The amount of [3H]glucose trapped
on each filter was used to calculate the wetting volume and was subtracted from the total Ca2+ bound to the filter, to give the specific amount of Ca2+ bound to the ATPase A correction was also applied for nonspecific binding of Ca2+to the lipid [13]
Modeling of the 2-APB-binding site on the Ca2+-ATPase Molecular graphics and docking procedures were per-formed principally with the SYBYL V6.5 package (Tripos Inc) A Silicon Graphics Octane 2 workstation was used for all graphics and calculations The drug 2-APB was initially sketched inSYBYLand then subjected to geometry optimization with GAUSSIAN98 (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 Ca2+-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 fromAMBER4.1 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 theVMDandRASTER3D
software packages [29]
Trang 4R E S U L T S
Inhibition of Ca2+-ATPase activity and Ca2+uptake
Figure 1 shows the effects of 2-APB on Ca2+-dependent
ATPase activity, using the phosphate liberation method As
can be seen from Fig 1A,B, the Ca2+-ATPase activity
in cerebellar microsomes is inhibited with an IC50 of
325 ± 19 lM However, the IC50 for SR Ca2+-ATPase
activity under the same conditions is 720 ± 45 lM 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 Co2+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 Co2+ions in the presence of
2-APB (i.e 500 lM 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 Ca2+ATPase Figure 3 shows the inhibition of purified Ca2+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
IC50of 2-APB at pH 7.2 was 800 ± 100 lM, however, at
pH 6.0 the IC50 was 70 lM This represents a 12-fold change in IC50between pH 6.0 and 7.2
Experiments were then performed to see how 2-APB affected ATPase activity at pH 7.2 as a function of Ca2+, ATP and Mg2+ Figure 3B shows the effects of Ca2+on ATPase activity, in the absence and presence of 800 lM
2-APB The data were fitted to the characteristic bell-shaped curve of Ca2+-dependent ATPase activity In the absence of 2-APB the ATPase had a Vmaxof 11.4 ± 0.3 UÆmg)1, with the Kmfor the stimulatory phase of 0.40 ± 0.03 lM, and the Km for the inhibitory phase of 0.35 ± 0.06 mM However, in the presence of 2-APB (800 lM), the Vmax was reduced to 5.9 ± 0.2 UÆmg)1, and stimulatory and inhibitory Km values increased to 0.90 ± 0.03 and 0.77 ± 0.33 mM, respectively These results therefore sug-gest that 2-APB may affect Ca2+binding
Figure 3Cshows 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 catalytic site is where ATP binds and phosphorylates the ATPase, while the low affinity
regulatory site 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 Km was 9.4 ± 1.6 lM, with a Vmax of
Fig 1 Effects of 2-APB on Ca2+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 , pH 7.0 Each data point is the
mean ± SD of three determinations.
Fig 2 2-APB increases membrane permeability The trace represents experiments of Co2+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.
Trang 55.4 ± 0.3 UÆmg)1and the regulatory Kmwas 1.3 ± 0.5 mM
with a corresponding Vmax of 8.1 ± 1.0 UÆmg)1 In the
presence of 2-APB (800 lM), the data could be fitted
assuming, the Km for both catalytic and regulatory sites
were unchanged (i.e 9.4 ± 2.8 and 1.3 ± 0.5 mM,
respec-tively) The Vmax values, however, were reduced The
catalytic and regulatory Vmaxvalues were 2.5 ± 0.1 and
5.4 ± 0.4 UÆmg)1, respectively Therefore 2-APB appeared
to have no effect on the apparent Kmfor 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 Mg2+ Mg2+ inhibits ATPase
activity at high concentrations with an IC50value of 8 mM,
which is not changed in the presence of 800 lM 2-APB
Again, the Vmaxis decreased from 14.3to 7.2 UÆmg)1(taken
at the optimal [Mg2+] of 2.5 mM) Therefore, 2-APB is
unlikely to have any effect on Mg2+binding to the ATPase
The effects of tryptophan fluorescence changes
associated with Ca2+binding
To assess whether 2-APB has an effect on the conformational
changes associated with Ca2+ binding to the ATPase,
tryptophan fluorescence was monitored in the absence and
presence of 2-APB at varying free Ca2+concentrations The change in tryptophan fluorescence induced by Ca2+has been attributed to a change in E1 conformational states during the process of Ca2+ binding [32] Figure 4A,B illustrates the change in tryptophan fluorescence induced by Ca2+in the presence and absence of 2-APB both at pH 6.0 and
pH 7.2 In all results, the DFmaxvalues did not significantly change (i.e 9.7–10.1% DFmaxat both pH values) There was, however, a decrease in the EC50values At pH 7.2, in the absence of 2-APB, the EC50 was 0.6 ± 0.1 lM Upon addition of 3 mM2-APB, this value increased threefold to 1.7 ± 0.4 lM At pH 6.0, the difference was even more dramatic as the EC50value changed from 11.5 ± 0.1 lMin the absence of 2-APB to 100 ± 11 and 350 ± 10 lMin the presence of 300 lM and 3 mM 2-APB, respectively These results therefore indicate that 2-APB affects the conforma-tional changes associated with Ca2+binding to the Ca2+ ATPase and that these effects are much greater at a lower pH Measuring45Ca2+binding to the ATPase
To deduce whether 2-APB was directly affecting Ca2+ binding,45Ca2+binding experiments were also performed
on the purified ATPase (Fig 5) The binding curves fitted to the data in Fig 5 give similar B values in the absence and
Fig 3 Effects of 2-APB on the purified skeletal muscle Ca2+ATPase activity as a function of free [Ca2+], [ATP] and [Mg2+] Activities of the Ca2+ 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 [Ca2+] (B); [ATP] (C ) and [Mg2+] (D), measured at pH 7.2, 37 Cin the absence (j) or presence (s) of 800 l M , 2-APB Each data point is the mean ± SD of three to four determinations.
Trang 6presence of 2-APB (20 ± 2 nmolÆmg)1in the absence of
2-APB and 21 ± 4 nmolÆmg)1 the presence of 3 mM
2-APB) The Kdfor Ca2+binding, although, were
substan-tially altered, as in the absence of 2-APB the Kd was
0.4 ± 0.2 lM, while in the presence of 3 mM 2-APB this
increased almost 20-fold to 6.9 ± 0.9 lM In addition, The
cooperativity of Ca2+ 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 mM 2-APB These results demonstrate that
2-APB inhibits Ca2+ binding to the Ca2+ATPase in a
competitive manner, making it noncooperative in the
process
Kinetics of conformational changes associated
with Ca2+binding and dissociation to the Ca2+-ATPase
The rate constants for the conformational changes
associ-ated with either Ca2+binding or Ca2+dissociation to the
ATPase were measured in the absence and presence 2-APB (3 mM) 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 R2 values‡ 0.9) In Table 1, it can be seen that the rate constant associated with Ca2+binding is decreased quite dramatically in the presence of 2-APB (by nearly eightfold)
In addition, the rate constant for Ca2+dissociation in the presence of 2-APB is also substantially increased by nearly fourfold As the Kdfor any binding process is related to the ratio of koff/kon, a decrease in the rate constant for
Ca2+binding, and an increase in the rate constant for Ca2+ dissociation will lead to a decreased affinity for Ca2+
binding as shown earlier
E2fi E1 transition of the ATPase
To determine whether 2-APB affects the E2fi E1 transi-tion of the ATPase, the fluorescence change induced by
Ca2+ on FITC-labelled Ca2+ ATPase was measured at
pH 6 Due to the effects of 2-APB on Ca2+binding, 1 mM
Ca2+was added to ensure the complete transition from the E2 and E1 step As can be seen in Fig 7, 2-APB caused a decrease in the Ca2+-dependent FITC-ATPase fluorescence change In addition, the increase in fluorescence in going from E1 to E2, due to the addition of 400 lM orthovana-date, was also measured The fluorescence increase associ-ated with the addition of orthovanadate changed in the presence of 3 mM2-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 IC50for the Ca2+-induced fluorescence changes
of the FITC-ATPase was calculated to be 1.5 mM, while the
IC50for ATPase inhibition at this pH is considerably less (i.e 70 lM), 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
a function of free [Ca2+] 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 [Ca2+] 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) or 3 m M 2-APB (s) Each data point represents
the mean ± SD of three or four determinations.
Fig 5 Effects of 2-APB on45Ca2+binding to the purified ATPase Binding of45Ca2+to purified ATPase was measured as a function of free Ca2+, in the absence (j) and presence (s) of 3 m M 2-APB, at
25 C, pH 7.2 Each data point is the mean ± SD of three to five determinations.
Trang 7ATP binding and phosphorylation of the Ca2+-ATPase
Figure 8A shows the effects of 3 mM 2-APB on the
phosphorylation of the Ca2+ ATPase in SR The EC50
and maximum level of phosphoenzyme formation (E–Pmax)
in the absence of 2-APB was 11 ± 6 lM ATP and
1.5 ± 0.2 nmolÆmg)1ATPase, respectively In the presence
of 3 mM 2-APB, the EC50 and E–Pmaxwere affected (i.e
19 ± 6 lMATP 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 mM 2-APB (i.e apparent
Kd¼ 3.5 lMin 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 Ca2+-ATPase activity Upon initial analysis involving docking of 2-APB to the
Ca2+-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 Ca2+-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 Ca2+-dependent ATPase activity was measured in these microsomes in the presence and absence of 800 lM2-APB (Fig 9) In the wild-type enzyme, 800 lM 2-APB inhibited Ca2+-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 lM 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 Ca2+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 Ca2+ion binding and dissociation These values are presented as means ± SEM Data presented is a result of an average of 10–12 individual experiments.
Fig 7 Effects of 2-APB on the E2 to E1 conformationalstep The fluorescence change of FITC-labelled ATPase, induced by 1 m M
Ca2+, was measured as a function of 2-APB concentration, at 25 C,
pH 6.0.
Trang 8indicates a crucial role for the hydroxyl group of this
tyrosine residue in the binding of 2-APB to the enzyme
D I S C U S S I O N
2-APB has been used extensively recently to investigate the
effects of InsP3-induced Ca2+release and Ca2+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 [Ca2+] unrelated to its effects on
InsP3-sensitive Ca2+ channels and store-operated Ca2+
entry These effects occur when 2-APB is used at
concen-trations above 200 lM As mentioned previously, we have
shown that InsP3-induced Ca2+release from type 1 InsP3
receptors is also affected at similar concentrations [6]
Furthermore, research into store-operated Ca2+entry has
shown 2-APB to be an effective inhibitor at concentrations
of 10–100 lM [7–10] We therefore advise caution when interpreting results obtained with 2-APB, when it is used at concentrations above 200 lM, on C a2+signalling processes
As described in this study, 2-APB reduces the affinity for
Ca2+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 (NH3+) 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
Ca2+-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 (Ca2+ 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 pKa for 2-APB when bound to the Ca2+-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
Ca2+-binding sites (site I: T799, E771, N768, E908, D800; site II: N796, A305, V304, E309, I307) and proposed the
Ca2+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 Ca2+ATPase at varying concentrations of
[c- 32 P]ATP, in the absence (j) and presence (s) of 3 m M 2-APB (B)
shows the spectroscopic change attributed to TNP-ADP binding to the
ATPase in the absence (j) and presence (s) of 3 m M 2-APB,
mea-sured by dual wavelength spectroscopy, at wavelengths 390 nm and
422 nm The experiments were performed at 25 C , pH 7.2 Each data
point represents the mean ± SD of three to five determinations.
Fig 9 Effects of 2-APB on Ca 2+ dependent ATPase activity of SR
Ca2+ 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 , pH 7.2 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)1Æmg)1.
Trang 9responsible 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 Ca2+ ATPase is in a
vanadate-bound (E2) state Furthermore, mutational
experiments involving the L6–7 loop have revealed a
decrease in Ca2+-dependent ATPase activity, with some
mutants also inhibiting Ca2+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 Ca2+ 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 Ca2+-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 Ca2+-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 Ca2+-binding site and plays a
crucial role in interacting with the Ca2+ions at both binding
sites [18] This may therefore account for the effects of
2-APB on Ca2+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 Ca2+-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 channel 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
Ca2+binding and ATP-dependent phosphorylation of the ATPase
The fact that 2-APB decreases Ca2+ binding by both reducing the rate constant for binding as well as increasing the rate constant for C a2+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 Ca2+-ATPase that reduces its affinity for Ca2+ 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 Ca2+-binding sites
A C K N O W L E D G E M E N T S
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 BBSRCfor a PhD studentship to J G B., the BHF for a PhD studentship to L L W., the MRCfor the bioinformatics grant (64600017) and Dr Shahidul Islam for encouragement to undertake this study.
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