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Effects of High-Affinity Inhibitors on Partial Reactions, Charge Movements, and Conformational States of the Ca²⁺ Transport ATPase (Sarco-Endoplasmic Reticulum Ca²⁺ ATPase)

Department of Chemistry, University of Florence, Sesto Fiorentino, Italy (F.T.-B., G.B., M.R.M.); Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel (D.M.T.); and California Pacific Medical Center Research Institute, San Francisco, California (D.L., G.I.)

Received November 22, 2007; accepted January 22, 2008

	Abstract

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The inhibitory effects of thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone, and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene on the Ca²⁺ ATPase were characterized by comparative measurements of sequential reactions of the catalytic and transport cycle, including biochemical measurements and detection of charge movements within a single cycle. In addition, patterns of ATPase proteolytic digestion with proteinase K were derived to follow conformational changes through the cycle or after inhibitor binding. We find that thapsigargin, cyclopiazonic acid, and 2,5-di(tert-butyl)hydroquinone inhibit Ca²⁺ binding and catalytic activation as demonstrated with isotopic tracers and lack of charge movement upon addition of Ca²⁺ in the absence of ATP. It has been shown previously that binding of these inhibitors requires the E2 conformational state of the ATPase, obtained in the absence of Ca²⁺. We demonstrate here that E2 state conformational features are in fact induced by these inhibitors on the ATPase even in the presence of Ca²⁺. The resulting dead-end complex interferes with progress of the catalytic and transport cycle. Inhibition by 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene, on the other hand, is related to interference with a conformational transition of the phosphorylated intermediate (E1P · 2Ca²⁺ to E2-P · 2Ca²⁺ transition), as demonstrated by increased phosphoenzyme levels and absence of bound Ca²⁺ translocation upon addition of ATP. This transition includes large movements of ATPase headpiece domains and transmembrane segments, produced through utilization of ATP-free energy as the "conformational work" of the pump. We conclude that the mechanism of high-affinity Ca²⁺ ATPase inhibitors is based on global effects on protein conformation that interfere with ATPase cycling.

Drug interactions with SERCA first came to light when it was discovered that thapsigargin (TG), a plant-derived sesquiterpene lactone (Fig. 1) (Rasmussen et al., 1978; Christensen et al., 1982), produces total and specific Ca²⁺ ATPase inactivation at extremely low concentrations (Lytton et al., 1991; Sagara and Inesi, 1991). TG has become a very useful tool for experimental manipulations of Ca²⁺ signaling in cells (Hussain and Inesi, 1999) and is presently considered for possible therapeutic applications (Denmeade and Isaacs, 2005; Søhoel et al., 2006). After the discovery of the TG effect, other compounds, such as cyclopiazonic acid (CPA) (Goeger et al., 1988; Seidler et al., 1989), 2,5-di(tert-butyl)hydroquinone (DBHQ) (Moore et al., 1987; Murphy et al., 1992), and 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene (TITU) (Berman and Karlish, 2003; Hua et al., 2005), were found to inhibit SERCA (Fig. 1).

View larger version (9K): [in this window] [in a new window]   Fig. 1. Structures of inhibitors: TG (A), CPA (B), DBHQ (C), and TITU (D).

An interesting effect of TG is stabilization of ATPase molecules in ordered arrays (Sagara et al., 1992), thereby facilitating diffraction studies of protein structure. In fact, crystallization of ATPase has been obtained in the absence of Ca²⁺ and in the presence of TG (Toyoshima and Nomura, 2002), demonstrating the TG binding site within a cavity surrounded by the M3, M5, and M7 transmembrane helices near the cytosolic side of the membrane bound region, as also indicated by mutational studies (Zhong and Inesi, 1998; Yu et al., 1999). Crystallographic evidence of CPA and DBHQ bound to SERCA was subsequently obtained, revealing distinct binding sites located near the cytosolic side of the membrane-bound region. CPA resides near the access to the Ca²⁺ sites, locking M1 and M2 against M4 (Moncoq et al., 2007; Takahashi et al., 2007), whereas DBHQ binding is favored by engagement of hydroxyl groups in hydrogen bonding with Asp59 (M1) and Pro308 (M4) and by hydrophobic contacts of butyl groups with neighboring nonpolar residues (Obara et al., 2005). Note that, in all cases, crystallization of ATPase with bound TG, CPA, or DBHQ required a Ca²⁺-free medium. Crystallization of ATPase in the presence of TITU was never obtained.

With the experiments reported here, we compared systematically the effects of these inhibitors on sequential steps of the catalytic cycle revealed by biochemical and electrical measurements, with conformational effects revealed by changes in exposure of ATPase proteolytic sites to proteinase K in the native membrane environment. In particular, we endeavored to clarify whether long-range effects on protein conformation play an important role in the inhibitory mechanisms of these compounds.

	Materials and Methods

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Reagents. Calcium, potassium, sodium, magnesium chloride, and MOPS were obtained from Merck (Whitehouse Station, NJ) at analytical grade. ATP disodium salt (97%) and dithiothreitol (DTT, 99% purity) were purchased from Fluka (Buchs, Switzerland). Octadecanethiol (98%) from Sigma-Aldrich (St. Louis, MO) was used without further purification. Choline chloride, EGTA, and calcimycin (calcium ionophore A23187 [GenBank] ) were obtained from Sigma-Aldrich. Potassium oxalate, lithium dodecyl sulfate, sucrose, β-mercaptoethanol, bromphenol blue, and TG were purchased from Sigma-Aldrich. CPA and DBHQ were obtained from BIOMOL Research Laboratories (Exeter, UK). Br-TITU and Br₂-TITU (1.65:1 ratio) were synthesized according to the method of Tal and Karlish (1995). Diphytanoylphosphatidylcholine was purchased from Avanti Polar Lipids (Alabaster, AL) and then solubilized (7.5 mg/ml) in n-decane (Merck).

ATPase Preparation. Sarcoplasmic reticulum vesicles were obtained by extraction from the fast-twitch hind-leg muscle of a New Zealand White rabbit, followed by homogenization and differential centrifugation as described by Eletr and Inesi (1972). The vesicles so obtained (light vesicles), derived from longitudinal SR membrane, contained only negligible amounts of the ryanodine receptor Ca²⁺ channel associated with junctional SR.

Functional Measurements. ATPase activity was measured at 25°C in a reaction mixture containing 30 µg/ml SR protein, 50 mM MOPS, pH 7, 50 mM KCl, 3 mM MgCl₂, 1 µg of A23187 [GenBank] ionophore, and 2 mM EGTA in the presence or absence of 2 mM CaCl₂. The reaction was started by addition of 2 mM ATP, and samples were taken at serial times for Pi determination.

Ca²⁺ binding to the ATPase in the absence of ATP was measured by incubating SR vesicles (40 µg/ml) in 20 mM MOPS, pH 7.0, 80 mM KCl, 5 mM MgCl₂, and 10 µM [⁴⁵Ca]CaCl₂. After 5 min of incubation at 25°C, the vesicles were loaded onto 0.45-µm Millipore filters by vacuum suction. The filters were then collected, blotted, and processed for determination of radioactivity.

ATP-dependent Ca²⁺ transport by SERCA was measured at 25°C in a reaction mixture containing 50 mM MOPS, pH 7, 80 mM KCl, 2 mM MgCl₂, 50 µg/ml microsomal protein, 5 mM potassium oxalate, and 10 µM free Ca²⁺ with ⁴⁵Ca tracer. ATP (1 mM) was added to start the reaction, and, at various times a 1-ml reaction mixture was loaded onto a 0.45-µm filter (Millipore, Billerica, MA) by vacuum suction and washed with 15 ml of 2 mM LaCl₃ and 10 mM MOPS, pH 7.0. The filter was then processed for determination of radioactivity by scintillation counting.

Enzyme phosphorylation by ATP was measured in an ice-cold reaction mixture containing 50 mM MOPS, pH 7.0, 80 mM KCl, 2 mM MgCl₂, 10 µM free Ca²⁺, and 50 µg/ml microsomal protein. The reaction was started by the addition of 10 µM [-³²P]ATP and quenched at various times with 1 M perchloric acid. The quenched samples were then loaded onto 0.45-µm Millipore filters by vacuum suction and washed with 15 ml of 0.1 M perchloric acid and 5 ml of cold water. The filters were then processed for determination of radioactivity by scintillation counting.

Measurement of Charge Movements. Charge movements were measured by adsorbing the SR vesicles containing the Ca²⁺-ATPase onto a mixed alkanethiol/phospholipid bilayer anchored to a gold electrode [the so-called solid supported membrane (SSM)]. The SSM consisted of an octadecanethiol monolayer covalently linked to the gold surface via the sulfur atom with a diphytanoylphosphatidylcholine monolayer on top of it (Pintschovius and Fendler, 1999; Tadini-Buoninsegni et al., 2004).

SR vesicles, after a brief sonication in the absence of detergent, were first adsorbed on the SSM, and the protein was then activated by the rapid injection of a solution containing a suitable substrate (e.g., Ca²⁺ or ATP). If at least one electrogenic step (i.e., a net charge movement across the vesicular membrane generated by the protein) is involved in the relaxation process that follows protein activation, a current transient can be recorded by the SSM method (Tadini-Buoninsegni et al., 2006). In particular, the electrical response of the ion pump can be monitored under potentiostatic conditions. In this case, movement of a net charge across the activated protein is compensated for by a flow of electrons along the external circuit to keep the applied voltage V constant across the whole metal/solution interphase. The resulting current transient is recorded as a function of time. Normally, experiments are carried out under short circuit conditions (i.e., at zero applied voltage relative to the reference electrode). It should be pointed out that the SSM technique detects pre-steady-state current transients within the first catalytic and transport cycle and is not sensitive to stationary currents after the first cycle. Useful information is gained from current transients. In fact, numerical integration of each transient is related to a net charge movement, which depends upon the particular electrogenic event (i.e., after Ca²⁺ or ATP concentration jumps). In addition, kinetic information can be obtained by fitting the current versus time curves to a sum of exponentially decaying terms. Recently, the traditional SSM method has been robotized and has become commercially available (SURFE²R; IonGate Biosciences GmbH, Frankfurt am Main, Germany).

View larger version (10K): [in this window] [in a new window]   Fig. 2. Effects of various inhibitors on steady-state Ca2+-ATPase activity.

In the experiments with the different inhibitors, the drug was added at the same concentration to both solutions from a stock solution in dimethyl sulfoxide. The concentration-jump experiments have been carried out by using the SURFE²R^One device. The SSM sensor, the experimental setup, and the solution exchange technique are described in Kelety et al. (2006).

To verify the reproducibility of the current transients generated within the same set of measurements on the same SSM, each single measurement of the set was repeated 4 to 5 times and then averaged to improve the signal-to-noise ratio. Average standard deviations were usually found to be no more than ±5%.

Free Ca²⁺ concentration was calculated with the computer program WinMAXC (http://www.stanford.edu/~cpatton/winmaxc2.html). Unless otherwise stated, 1 µM A23187 [GenBank] , the calcium ionophore, was used to prevent formation of a Ca²⁺ concentration gradient across the SR vesicles. The temperature was maintained at 22-23°C for all the experiments.

Limited proteolytic digestion was performed in reaction mixtures containing 50 mM MOPS, pH 7.0, 50 mM NaCl, 2.0 mM MgCl₂, 0.05 mg/ml SR microsomal protein, and 0.05 mg of proteinase K. CaCl₂ and EGTA were added as indicated in the figures. After incubation at 25°C for various time intervals, the reaction was quenched with trichloroacetic acid (2.5%), and the protein was solubilized with a medium containing lithium dodecyl sulfate (1%), MOPS (0.312 M), pH 6.8, sucrose (3.75%), β-mercaptoethanol (1.25 mM), and bromphenol blue (0.025%). The samples were then subjected to electrophoretic analysis on 12% gels, and the protein bands were stained with Coomassie Blue R-250. Alternatively, Western blots were obtained using the monoclonal antibody MA3911 or MA3912 (Affinity BioReagents, Golden, CO), followed by goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibodies and visualization with an enhanced chemiluminescence-linked detection system (Pierce, Rockford, IL). The MA3911 antibody reacts preferentially with the amino-terminal region of the ATPase, whereas the MA3912 reacts preferentially with the carboxy-terminal region.

	Results

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Measurements of ATPase activity were first conducted in the presence of the Ca²⁺ ionophore A23187 [GenBank] to produce passive leak of transported Ca²⁺, thereby preventing back inhibition by high [Ca²⁺] buildup in the lumen of the SR vesicles. Linear ATP hydrolysis is obtained under these conditions as a function of time, yielding reliable steady-state rates of ATPase activity in the presence of saturating concentrations of Ca²⁺ and ATP. A comparative evaluation of the concentrations required for inhibition of the Ca²⁺-ATPase activity by various inhibitors is shown in Fig. 2. As previously reported, the K_i,app values vary from the subnanomolar range for TG to 0.05 µM for CPA, 0.48 µM for DBHQ, and 15 µM for TITU (Table 1). It is noteworthy that these are apparent values and may not correspond exactly to the dissociation constants (K_d) of these compounds from the ATPase protein. Steady-state measurement of ATP-dependent Ca²⁺ transport (in the presence of oxalate to obtain linear activity) revealed a pattern of inhibition by TG, CPA, DBHQ, and TITU quite similar to that observed by measurements of ATPase (not shown).

Considering the sequence of partial reactions comprising the catalytic and transport cycle, it was previously observed that TG, CPA, and DBHQ interfere with enzyme activation by Ca²⁺, thereby preventing ATP utilization and formation of phosphoenzyme intermediate (Table 2). On the other hand, TITU allows Ca²⁺ binding and ATP utilization for formation of phosphoenzyme intermediate (E1P). In fact, in the presence of TITU, phosphorylated enzyme intermediate is formed and rises to nearly match the enzyme stoichiometry. However, its hydrolytic cleavage is sharply reduced (Table 2), and translocation of bound Ca²⁺ does not occur.

An informative method for further characterization of Ca²⁺ transport coupled to ATPase activity is measurement of pre-steady-state charge movements within a single Ca²⁺-ATPase cycle. The measurements are obtained with vesicular fragments of SR membrane adsorbed on a SSM and subjected to Ca²⁺ jumps in the absence of ATP or to ATP jumps in the presence of Ca²⁺. Current transients induced by addition of 10 µM Ca²⁺ and subsequent addition of 100 µM ATP in the presence of 10 µM Ca²⁺ are shown in Fig. 3. In particular, curve (a) in Fig. 3 represents the initial binding of Ca²⁺ to the ATPase transport sites in the absence of ATP. Curve (b) in Fig. 3 corresponds to the displacement of the bound Ca²⁺ through the ATPase molecule upon utilization of ATP. Each electrogenic event has its own time constant, as shown by the different time frames of the two current transients. Therefore, the charge obtained by numerical integration of each transient is attributed to these sequential electrogenic events, that is, binding of Ca²⁺ to the cytoplasmic sites (Fig. 3, curve a) and vectorial translocation of bound Ca²⁺ after utilization of ATP (Fig. 3, curve b) (Tadini-Buoninsegni et al., 2006). It should be understood that this technique detects single electrogenic steps within the first catalytic and transport cycle and is not sensitive to steady-state events after the first cycle.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Current transients induced by a 10 µM free Ca2+ concentration jump in the absence of ATP (a) and a 100 µM ATP concentration jump in the presence of 10 µM free Ca2+ (b). i, current; t, time.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Dependence of the normalized charge after 10 µM free Ca2+ concentration jumps on inhibitor concentration: TG (), CPA (), DBHQ (), and TITU (). In each series, the charges are normalized with respect to the maximum charge observed in the absence of the corresponding inhibitor. The solid curves were obtained by fitting with a sigmoid function the normalized charge.

View larger version (10K): [in this window] [in a new window]   Fig. 5. Dependence of the normalized charge after 100 µM ATP concentration jumps in the presence of 10 µM free Ca2+ on inhibitor concentration: TG (), CPA (), DBHQ (), and TITU (). In each series, the charges are normalized with respect to the maximum charge observed in the absence of the corresponding inhibitor. The solid curves were obtained by fitting with a sigmoid function the normalized charge.

View larger version (37K): [in this window] [in a new window]   Fig. 6. a, crystallographic models of the Ca2+ ATPase in the E2 · TG (left, Protein Data Base code 1iwo) and in the E1 · 2Ca2+ (right, Protein Data Base code 1su4 [PDB] ). The sites for initial digestion by proteinase K are in the loops intervening between the A domain and the M2 and M3 transmembrane segments. Note their displacement as the A domain rotates as a consequence of the E1 to E2 transition. Digestion patterns of ATPase with proteinase K in the absence (b) and in the presence of Ca2+ (c). The first lane on the left shows a sample with proteinase K denatured before addition of ATPase. The E1 pattern yields complementary 83-kDa (carboxyl terminus) and 28-kDa (amino terminus) bands, whereas the E2 pattern yields additional 95-kDa (carboxyl terminus) and 14-kDa (amino terminus) bands. Note that the E2 pattern is obtained even in the presence of Ca2+ when TG (1.0 µM), CPA (10 µM), or DBHQ (20 µM) are added to the reaction mixture, whereas the E1 pattern is obtained in the presence of TITU (100 µM). Digestion (30 min at 25°C) and electrophoresis as explained under Materials and Methods. The free Ca2+ concentration, when present, was 50 µM. Total protein loaded per well was 30 µg. SERCA protein and fragments thereof were evidenced by Western blotting with specific antibodies. digest., digestion.

	Discussion

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The discovery of SERCA inhibitors has opened favorable avenues for mechanistic studies on Ca²⁺ homeostasis (Hussain and Inesi, 1999), stereochemistry of drug interactions with proteins (Logan-Smith et al., 2002; Wootton and Michelangeli, 2006; Lape et al., 2008), and possible therapeutic applications (Eckstein-Ludwig et al., 2003; Denmeade and Isaacs, 2005; Søhoel et al., 2006). With the experiments reported here, we endeavored to obtain a comparative characterization of the interactions of four inhibitors with SERCA, their interference with partial reactions of the catalytic and transport cycle, and the relation of inhibitory mechanisms to protein conformation. In addition to well established methods of functional characterization, in these experiments we have used a novel method to measure charge movements, thereby detecting separately Ca²⁺ binding to the transport sites in the absence of ATP and vectorial translocation of bound Ca²⁺ upon addition of ATP, as sequential electrogenic steps within a single ATPase cycle. In addition, we followed the pattern of ATPase cleavage by proteinase K, thereby determining the conformational state induced by each inhibitor on the ATPase within the native membrane environment.

In accordance with previous reports, we find that TG, CPA, DBHQ, and TITU inhibit SERCA within the nanomolar to micromolar range (Table 1). Both biochemical and electrical measurements demonstrate that TG, CPA, and DBHQ interfere with Ca²⁺ binding (Table 1; Fig. 4), whereby lack of catalytic activation prevents formation of phosphorylated enzyme intermediate (Table 2; Scheme 1). On the contrary, Ca²⁺ binding, catalytic activation, and formation of phosphoenzyme intermediate occur normally in the presence of TITU (Figs. 2 and 4; Table 2). High levels of phosphoenzyme (Table 2), inhibition of ATP-dependent charge movements (Fig. 5), and low cleavage of phosphoenzyme (E1P) obtained by utilization of ATP are prominent features of TITU inhibition. This, in conjunction with normal cleavage of phosphoenzyme (E2-P) obtained by utilization of Pi (Hua et al., 2005), demonstrates that TITU inhibits the E1P · 2Ca²⁺ to E2-P · 2Ca²⁺ transition (Scheme 1).

View larger version (11K): [in this window] [in a new window]   Scheme 1. Sequence of partial reactions in the catalytic and transport cycle of the Ca2+ ATPase.

We found that, in the presence of TG, CPA, and DBHQ, ATPase digestion with proteinase K yields an E2 pattern even in the presence of Ca²⁺. This is in agreement with the structures observed under conditions of crystallization in the absence of Ca²⁺ and in the presence of TG, CPA, or DBHQ (Toyoshima and Nomura, 2002; Obara et al., 2005; Moncoq et al., 2007; Takahashi et al., 2007). The crystallographic structures demonstrate that the E2 state favors binding of these inhibitors and define the binding sites. On the other hand, our experiments demonstrate that conformational features of the E2 state are produced in the native membrane environment by TG, CPA, and DBHQ (used separately), even in the presence of Ca²⁺ (Fig. 6, b and c). Therefore, it is clear that the dead-end complex formed by these specific inhibitors is related to a global conformational effect on the ATPase (Wictome et al., 1992; Logan-Smith et al., 2002) whereby catalytic cycling is prevented. It is noteworthy that the conformational state stabilized by the inhibitors is quite similar, but not identical, to the E2 ground state of the physiological ATPase cycle. In fact, local and diverse interactions occur at each inhibitor's binding site. From the functional point of view, lack of reactivity to catalytic ligands and substrate is the most prominent feature distinguishing the dead-end complex from the physiological E2 state.

Contrary to the effects of TG, CPA, and DBHQ, digestion with TITU clearly yields the E1 pattern in the presence of Ca²⁺ (Fig. 6, b and c). This is in agreement with the observation that TITU does not interfere with Ca²⁺ binding, as demonstrated directly with isotopic tracer (Table 2) and by measurements of Ca²⁺-induced charge movements (Fig. 4). On the other hand, turnover of the phosphoenzyme obtained by utilization of ATP and translocation of bound Ca²⁺ are inhibited by TITU, as demonstrated with isotopic tracer and by measurements of ATP-induced charge movements (Fig. 5). Accumulation of high E1P · 2Ca²⁺ steady-state levels, in conjunction with normal hydrolytic cleavage of the phosphoenzyme obtained by utilization of Pi (Hua et al., 2005), indicates that the E1P · 2Ca²⁺ to E2-P · 2Ca²⁺ transition is inhibited by TITU (Scheme 1).

It was reported that, in addition to the Ca²⁺ ATPase, TITU also inhibits the Na⁺/K⁺ ATPase (Hoving et al., 1995). In the Na⁺/K⁺ ATPase the mechanism of inhibition is rather complex, including stabilization of the E1 state at low concentrations and stabilization of the E2 state at higher concentrations. On the other hand, inhibition of the Ca²⁺ ATPase occurs prominently through stabilization of the E1 state (see above). It is then apparent that TITU is not strictly specific for the Ca²⁺ pump. It is noteworthy that ATPase crystals with TITU bound within the protein structure were never obtained. It is possible that TITU partitions within the membrane phase and then adheres to the membrane-bound enzyme protein, thereby interfering with large movements of the ATPase headpiece domains and transmembrane segments that are required for translocation of bound Ca²⁺ in the normal cycle. These movements, corresponding to the E1P · 2Ca²⁺ to E2-P transition (Toyoshima and Inesi, 2004), represent in fact the "conformational work" of the pump, whereby the free energy of ATP is utilized for reduction of the Ca²⁺ binding affinity and vectorial orientation of the Ca²⁺ binding sites (Inesi et al., 2008). Therefore, conceptually, TITU has a very interesting inhibitory mechanism, which could be referred to as "a stick in the turning wheel of the engine."

In conclusion, our experiments indicate that the mechanism of inhibition involves global effects on protein conformation, resulting in a dead-end complex of the Ca²⁺ ATPase with specific inhibitors such as TG, CPA, and DBHQ. The conformation of the complex is similar (but not identical) to the E2 state (Scheme 1), but its lack of reactivity to ligands and substrate interferes with progress of the catalytic and transport cycle. Inhibition by TITU, on the other hand, is related to interference with conformational transition of the phosphorylated intermediate, as required for energy transduction and active transport of bound Ca²⁺.

	Acknowledgements

 
We thank Serena Smeazzetto for preliminary SSM measurements on the effect of CPA on charge movements.

	Footnotes

 
This work was supported by the Ente Cassa di Risparmio di Firenze (PROMELAB project), the Ministero dell'Istruzione, dell'Università e della Ricerca (PRIN 2006), and the U.S. National Institutes of Health (R01-HL69830).

ABBREVIATIONS: TG, thapsigargin; CPA, cyclopiazonic acid; DBHQ, 2,5-di(tert-butyl)hydroquinone; TITU, 1,3-dibromo-2,4,6-tri(methylisothiouronium)benzene; SERCA, sarco-endoplasmic reticulum Ca²⁺ ATPase; SSM, solid supported membrane; MOPS, 3-(N-morpholino)propanesulfonic acid; SR, sarcoplasmic reticulum; DTT, dithiothreitol; conc., concentration.

Address correspondence to: Dr. Maria Rosa Moncelli, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. E-mail: moncelli{at}unifi.it

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