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: 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) 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. 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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;