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Originally published In Press as doi:10.1074/jbc.M510550200 on February 1, 2006

J. Biol. Chem., Vol. 281, Issue 14, 9547-9551, April 7, 2006
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Clotrimazole Inhibits the Ca2+-ATPase (SERCA) by Interfering with Ca2+ Binding and Favoring the E2 Conformation*

Gianluca Bartolommei{ddagger}, Francesco Tadini-Buoninsegni{ddagger}, Suming Hua§, Maria Rosa Moncelli{ddagger}, Giuseppe Inesi§, and Rolando Guidelli{ddagger}1

From the {ddagger}Department of Chemistry, University of Florence, 50019 Sesto Fiorentino, Florence, Italy and the §Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received for publication, September 27, 2005 , and in revised form, December 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clotrimazole (CLT) is an antimycotic imidazole derivative that is known to inhibit cytochrome P-450, ergosterol biosynthesis and proliferation of cells in culture, and to interfere with cellular Ca2+ homeostasis. We found that CLT inhibits the Ca2+-ATPase of rabbit fast-twitch skeletal muscle (SERCA1), and we characterized in detail the effect of CLT on this calcium transport ATPase. We used biochemical methods for characterization of the ATPase and its partial reactions, and we also performed measurements of charge movements following adsorption of sarcoplasmic reticulum vesicles containing the ATPase onto a gold-supported biomimetic membrane. CLT inhibits Ca2+-ATPase and Ca2+ transport with a KI of 35 µM. Ca2+ binding in the absence of ATP and phosphoenzyme formation by the utilization of ATP in the presence of Ca2+ are also inhibited within the same CLT concentration range. On the other hand, phosphoenzyme formation by utilization of Pi in the absence of Ca2+ is only minimally inhibited. It is concluded that CLT inhibits primarily Ca2+ binding and, consequently, the Ca2+-dependent reactions of the SERCA cycle. It is suggested that CLT resides within the membrane-bound region of the transport ATPase, thereby interfering with binding and the conformational effects of the activating cation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clotrimazole (CLT)2 is an antimycotic imidazole derivative widely used for the treatment of yeast infections. Like other drugs belonging to the same imidazole family (e.g. miconazole and ketoconazole), its anti-mycotic effect is because of inhibition of cytochrome P-450, an enzyme involved in ergosterol biosynthesis by the yeast (1, 2). It has also been shown that CLT inhibits cell proliferation of several normal and cancer cell lines in vitro (3). Interference with cellular Ca2+ homeostasis contributes to this inhibition; in particular, the action of CLT involves depletion of intracellular Ca2+-stores, also confirmed in Madin-Darby canine kidney cells, where CLT induces Ca2+ release from thapsigargin-sensitive Ca2+-stores (4).

It is reported that CLT affects the Ca2+ pump activity of SR vesicles isolated from rabbit heart muscle, producing inhibition of the Ca2+-dependent reactions of the ATPase (SERCA2) obtained from cardiac myocytes (5). With the experiments reported here, we found that, in fact, CLT inhibited the Ca2+-ATPase. We investigated the effect of CLT on the Ca2+-ATPase (SERCA1) associated with native membrane vesicles derived from rabbit fast-twitch skeletal muscle. Under optimal coupling conditions, the SERCA pump translocates two calcium ions per ATP molecule from the cytoplasm to the lumen of SR with a turnover rate of about 10 s-1, forming a 3 orders of magnitude Ca2+ gradient (6-9). Altered SERCA function is relevant to pathogenic mechanisms of several diseases (10-12). Furthermore, SERCA function can be affected by several compounds of possible pharmacological interest, such as thapsigargin (TG) (13), cyclopiazonic acid (14), curcumin (15, 16), 2,5-di-tert-butyl-1,4-dihydroxybenzene (17), and 1,3-dibromo-2,4,6-tris-(methylisothiouronium)benzene (18).

In addition to biochemical characterization of the ATPase and its partial reactions, we performed direct measurements of charge translocation following adsorption of SR vesicles containing ATPase onto a biomimetic membrane supported by a gold electrode and activation by rapid mixing with a suitable substrate (19). This technique has been used successfully in studies of electrogenic transport by several membrane proteins, such as Na+, K+-ATPase (20, 21), melibiose permease (22), and Na+/proline antiporter (23), including the SERCA pump (24, 25). We found that CLT interferes specifically with Ca2+ binding to SERCA even in the absence of ATP. For this reason, the Ca2+-dependent reactions of this enzyme are also inhibited.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Calcium and magnesium chlorides and MOPS were obtained from Merck at analytical grade. ATP disodium salt (~97%) and dithiothreitol (≥99%) were purchased from Fluka. Octadecanethiol (98%) from Aldrich was used without further purification. EGTA, calcimycin (calcium ionophore A23187 [GenBank] ), and 1-[o-chloro-{alpha},{alpha}-diphenyl-benzyl]-imidazole (clotrimazole) were obtained from Sigma. The lipid solution contained diphytanoylphosphatidylcholine (Avanti%20Polar%20Lipids">Avanti Polar Lipids) and octadecylamine (≥99.0%, Fluka) (60:1) and was prepared at a concentration of 1.5% (w/v) in n-decane (Merck), as described by Bamberg et al. (26).

ATPase Preparation—Sarcoplasmic reticulum vesicles were obtained by extraction from the fast-twitch hind leg muscle of New Zealand White rabbit followed by homogenization and differential centrifugation, as described by Eletr and Inesi (27). The vesicles derived from longitudinal SR membrane contained only negligible amounts of the ryanodine receptor Ca2+ channel associated with junctional SR (light vesicles).

Free Ca2+ concentration was calculated with the computer program WinMAXC (28). Unless otherwise stated, 1 µM calcium ionophore A23187 [GenBank] was used to prevent Ca2+ accumulation by the SR vesicles (29).

ATPase Activity—Ca2+-ATPase hydrolytic activity was determined following Pi production by a colorimetric method (30). The reaction mixtures contained 20 mM MOPS (pH 7.0), 80 mM KCl, 3 mM MgCl2, 0.2 mM EGTA, 0.2 mM CaCl2, 2 µg of SR protein/ml, and 3 µM A23187 [GenBank] ionophore and were preincubated with various concentrations of CLT for 30 min. The reaction was started by addition of 1 mM ATP. The incubation temperature was 37 °C.

45Ca2+ Binding—Ca2+ binding in the absence of ATP was measured by incubating SR vesicles for 30 min (0.1 mg/ml) in a pH 7.0 reaction mixture containing 20 mM MOPS, 80 mM KCl, 5 mM MgCl2,10 µM free [45Ca]CaCl2, and 3 µM A23187 [GenBank] ionophore with different concentrations of CLT. TG (2 µM) was added to half of the samples to provide controls exhibiting no specific Ca2+ binding because it was demonstrated previously that TG prevents specific Ca2+ binding (13). The reaction temperature was 25 °C. The reaction mixture was filtrated with an 0.45-µm filter, and radioactivity was measured by a liquid scintillation counter.

Phosphorylation by [{gamma}-32P]ATP—Enzyme phosphorylation by ATP was measured in a pH 7.0 ice-cold reaction mixture containing 20 mM MOPS, 80 mM KCl, 2 mM MgCl2, 10, 50, or 200 µM CaCl2, 0.1 mg of SR protein/ml, and 3 µM A23187 [GenBank] ionophore after a preincubation with various concentrations of CLT for 30 min. Individual samples (1 ml) were started by the addition of 20 µM [{gamma}-32P]ATP and quenched at 1 s with 1 M perchloric acid. After filtration and washing of the sample, radioactivity of the sample was measured with a liquid scintillation counter.

Phosphorylation by [32P]Pi—Enzyme phosphorylation with Pi was measured at 25 °C in a pH 6.2 reaction mixture containing 50 mM MES, 10 mM MgCl2, 2 mM EGTA, and 20% Me2SO after a 30-min preincubation with various concentrations of CLT in the presence of 1 mM [32P]Pi and 0.5 mg of SR protein/ml. The reaction was acid-quenched after a 10-min incubation by the addition of 1 M perchloric acid. The EP measurements were conducted using a filtration method.

Measurement of Charge Movements—Charge movements were measured by adsorbing the SR vesicles containing the Ca2+-ATPase onto a biomimetic membrane supported by a gold electrode (the so-called solid-supported membrane (SSM)). The SSM consisted of an octadecanethiol monolayer covalently bound to a gold surface via the sulfur atom with a phospholipid monolayer on top of it (31). After adsorption, usually carried out at an applied potential of +0.1 V, the protein was activated by a rapid injection of a solution containing the appropriate substrate, e.g. ATP or Ca2+ ions. If at least one electrogenic step is involved in the relaxation process, a current transient (pre-steady state) can be recorded along the external circuit. The acquisition of transients obtained under different experimental conditions, together with their subsequent elaboration, can provide important kinetic information about protein function and/or its modulation by drugs, peptides, and small soluble proteins. In particular, from a current transient, three main data can be extracted: the peak current, the translocated charge, and the decay time constants. The charge is obtained by numerical integration of the current transient, whereas the decay time constants are provided by a multiexponential fitting of the transient (19, 20, 24, 25). The time constants are strictly correlated with the rate constants of the reaction steps involved in the activation of the pump (32). The preparation of the gold electrodes and the whole experimental set-up as well as the solution exchange technique are described in detail by Tadini-Buoninsegni et al. (25).

For the activation experiments, the composition of the solutions was the following: (a) for Ca2+ concentration jumps, the non-activating solution contained 150 mM choline chloride, 25 mM MOPS (pH 7.0), 0.25 mM EGTA, 1 mM MgCl2, 0.2 mM dithiothreitol, and the activating solution had the same composition plus the required CaCl2 amount added from a 5, 50 or 500 mM stock solution; (b) for ATP concentration jumps, the non-activating solution contained 150 mM choline chloride, 25 mM MOPS (pH 7.0), 1 mM EGTA, 1 mM MgCl2, 1.1 mM CaCl2 (100 µM free Ca2+), and 0.2 mM dithiothreitol. The activating solution had the same composition plus 100 µM ATP. CLT was added from a 10-mM stock solution in Me2SO to the buffer solution when needed.


Figure 1
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  		FIGURE 1.
ATPase activity of Ca2+-ATPase determined in the presence of increasing concentration of CLT. Two values of calcium concentration were used: 10 µM (•) and 200 µM ({circ}).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We studied the effect of CLT on the SR Ca2+-ATPase by a series of biochemical measurements, including ATPase activity, [45Ca]Ca2+ binding in the absence of ATP, formation of the phosphorylated enzyme intermediate (E-P) by utilization of [{gamma}-32P]ATP in the presence of Ca2+, and formation of E-P by utilization of [32P]Pi in the absence of Ca2+. In addition, we performed pre-steady state activation experiments with the SSM-based technique (Ca2+ concentration jumps and ATP concentration jumps in the presence of CLT).

Biochemical Experiments—It is shown in Fig. 1 that the steady state hydrolytic activity of the Ca2+-ATPase (in the presence of 10 or 200 µM free Ca2+) is inhibited by CLT within the µM concentration range. It is noteworthy that measurements at two different Ca2+ concentrations (10 and 200 µM) yield the same dependence upon the inhibitor concentration, indicating that no protection is obtained by increasing the Ca2+ concentration above the saturating level.

With the aim of determining whether the effect of CLT is a total inhibition of SERCA activity (as that of TG) or is rather because of interference with a specific partial reaction, we measured independently [45Ca]Ca2+ binding in the absence of ATP, E-P formation by utilization of [{gamma}-32P]ATP in the presence of Ca2+, and E-P formation by utilization of [32P]Pi in the absence of Ca2+. It is shown in Fig. 2 that 45Ca2+ binding and E-P formation from [{gamma}-32P]ATP are inhibited with a similar pattern as the concentration of CLT is increased from zero to 50 µM. It should be pointed out that a limit in the solubility of CLT in the aqueous reaction medium renders a concentration increase above 50 µM rather ineffective. It is noteworthy that the same pattern of inhibition with respect to the CLT concentration was observed when enzyme phosphorylation with ATP was measured in the presence of 10, 50, or 200 µM Ca2+, indicating that the CLT inhibition is not competitive with respect to Ca2+. A most interesting observation is that E-P formation from [32P]Pi (in the absence of Ca2+) is only minimally reduced (Fig. 2), indicating that CLT inhibition of the SERCA pump is mainly exerted on Ca2+ binding and the Ca2+-dependent partial reactions of the enzymatic cycle.


Figure 2
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  		FIGURE 2.
45Ca2+ binding to the SERCA (•) and E-P formation from [{gamma}-32P]ATP ({circ}, {blacksquare}, {square}) and [32P]Pi ({blacktriangleup}) as determined at various CLT concentrations. The experiments were conducted as described under "Materials and Methods." The Ca2+ concentration for enzyme phosphorylation was 10 ({circ}), 50 ({blacksquare}), and 200 µM ({square}).

 
On and Off Ca2+ Concentration Jumps in the Presence of CLT in Pre-steady State—A Ca2+ concentration jump, performed with the SSM-based technique, yielded a current transient that could be ascribed to Ca2+ binding to the ATPase. An ON signal was obtained following the injection of the Ca2+-containing solution (Fig. 3A, step 1), and an OFF signal was obtained upon calcium release from the binding site following injection of a solution containing EGTA and no calcium (Fig. 3A, step 2) (25). We then performed these experiments in the presence of increasing concentrations of CLT to assess the effect of the drug on Ca2+ binding in the absence of ATP. In agreement with direct measurements of Ca2+ binding (Fig. 2), we found that charge movements attributed to Ca2+ binding were also reduced by CLT.

Fig. 4 compares a saturating calcium concentration jump in the absence and in the presence of 5 µM CLT, showing a clear reduction of the signal in the presence of the drug. Moreover, a titration with increasing Ca2+ concentrations (Fig. 5) shows that the signal is reduced to about one-third of that recorded in the absence of the inhibitor (25). On the other hand, despite the reduction in maximal binding, the Kd and n values (0.53 and 1.7 µM, respectively) obtained from fitting to the Hill function are very close to those obtained in the absence of drug, either by measurements of charge movements (25) or by direct measurements with a radioactive tracer (33). This indicates that non-saturating CLT reduces the amount of Ca2+ bound but does not affect the binding characteristics (i.e. affinity) of the enzyme molecules that are not totally inhibited.

We also found that titrations with increasing CLT concentrations in the presence of 10 or 100 µM Ca2+ concentrations (Fig. 6) yield the same inhibition curve. These results are in agreement with the experiments on enzyme phosphorylation with ATP (Fig. 2) and ATPase hydrolytic activity (Fig. 1) and imply that increasing the Ca2+ concentration above the saturating level does not protect the enzyme from CLT. Therefore, the CLT inhibition is not competitive with respect to Ca2+. On the other hand, we found that the effective CLT concentration is lower when the inhibitor is added before (KI = 7 µM, as in Fig. 6) rather than after Ca2+ (KI = 35 µM, as in Figs. 1, 2, and 7). A similarly greater resistance of the Ca2+-bound conformation of the enzyme (Ca2E1) as compared with the Ca2+-free conformation (E2) was found previously with respect to TG (13) and miconazole (34).

ATP Concentration Jumps in the Presence of CLT—An ATP concentration jump in the presence of Ca2+, performed on a SSM with SR vesicles containing Ca2+-ATPase adsorbed onto it, produces a current transient because of the electrogenicity of Ca2+ release into the lumen of the SR and, to some extent, to Ca2+ re-binding to the protein after the hydrolytic cleavage of P-E2 intermediate (Fig. 3B, steps 2 and 4). It was previously determined that the formation and hydrolytic cleavage of the phosphorylated intermediate are not electrogenic (35).


Figure 3
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  		FIGURE 3.
Reaction steps of the enzymatic cycle of SR Ca2+-ATPase. The gray boxes underline the protein starting conformations for a calcium concentration jump experiment (A) and for an ATP concentration jump experiment (B). Bold characters and arrows indicate an electrogenic event. The circled numbers point out the temporal sequence of the reaction steps during the experiment.

 


Figure 4
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  		FIGURE 4.
Comparison of saturating Ca2+ concentration jumps in the absence (solid line) or in the presence (dashed line) of 5 µM CLT. The charges associated with the current transients are 29.0 and 10.5 picocoulombs, respectively, corresponding to a 36% reduction of the signal in the presence of the drug. The total calcium amount is 0.25 mM, which results in a free concentration of 10.3 µM for the solution composition described under "Materials and Methods." i, current; t, time.

 
Fig. 7 shows the effect of 35 µM CLT on a 100 µM ATP concentration jump in the presence of saturating Ca2+ (100 µM). It is clear that the current transient because of Ca2+ translocation is affected by CLT. In fact, starting from no CLT and increasing CLT concentration up to 35 µM, peak currents are progressively decreased. Furthermore, integration of the current transients yields values for moved charges that exhibit a behavior very similar to that of peak currents with respect to the effect of CLT (Fig. 7, inset).

It is noteworthy that exponential fitting of the current transients yields two time constants for current decay. The two constants, {tau}1 and {tau}2, have average values of 27 ± 2 and 42 ± 3 ms, respectively and do not appear to be significantly affected by CLT (not shown), irrespective of the effect of CLT on peak currents and moved charge.


Figure 5
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  		FIGURE 5.
Titration of SERCA with calcium in the presence of 5 µM CLT obtained by Ca2+ concentration jumps experiments. Values are normalized with respect to the bound charge for a saturating Ca2+ concentration jump (10 µM) in the absence of CLT. The solid line represents the fitting to a Hill function (Q = Qnorm, max · Cn /(K0.5n + Cn)), which yields K0.5 = 0.53 ± 0.03 µM and n = 1.7 ± 0.2. The composition of the solution is indicated under "Materials and Methods."

 


Figure 6
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  		FIGURE 6.
Dependence of the normalized charge following Ca2+ concentration jumps on CLT concentration. Free calcium concentration was 10 µM (filled circle and solid line) or 100 µM (open circle and dashed line). The composition of the solution is indicated under "Materials and Methods."

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CLT is an antimycotic agent, the effect of which is attributed to inhibition of cytochrome P-450 and ergosterol biosynthesis in yeast (1, 2). It is also shown that CLT inhibits cell proliferation in vitro (3), interferes with calcium homeostasis in culture cells (3, 4), and inhibits the calcium pump (SERCA2) obtained with the microsomal fraction of heart muscle (5). We have characterized in detail the effect of CLT on the Ca2+-ATPase (SERCA1) activity associated with microsomal vesicles obtained from skeletal muscle. This enzyme preparation is highly purified and well suited to characterization of the catalytic cycle and its partial reactions (6-9). In addition, several SERCA inhibitors have been described (13-18), thereby yielding useful grounds for comparison of inhibitory mechanisms. Finally, the simultaneous use of biochemical and biophysical methods in the characterization of the CLT inhibitory mechanism has given us the opportunity to demonstrate clearly the functional relevance of charge transfer measurements and their correspondence to partial reactions of the ATPase cycle.

As outlined in Fig. 3B, the ATPase (SERCA) cycle requires initial activation through Ca2+ binding from the exterior of the vesicles (step 4) followed by utilization of ATP to form a phosphorylated intermediate (step 1). The bound Ca2+ is then dissociated into the lumen of the vesicles (Fig. 3B, step 2) followed by hydrolytic cleavage of the phosphoenzyme (step 3). Using direct binding assays by a radioactive tracer as well as measurements of charge movements by the SSM-based technique, we found that the initial Ca2+ binding, before utilization of ATP, is inhibited by CLT (Figs. 2, 4, and 6). It is of interest that plots of charge as a function of Ca2+ concentration (Fig. 5) yield affinity constants and cooperative behavior identical to that of equilibrium binding isotherms obtained in the absence of CLT by direct measurements with isotopic tracer (33). The correspondence of the two types of measurements is highly satisfactory and demonstrates the specificity of charge movement with regard to Ca2+ binding and its inhibition by CLT.


Figure 7
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  		FIGURE 7.
Current transients following 100µM ATP concentration jumps in the presence of 100 µM free calcium ions and in the absence (solid line) or in the presence (dashed line) of 35 µM CLT. The inset shows the normalized peak currents (•) and the translocated charges ({circ}) obtained from current transients following 100 µM ATP concentration jumps in the presence of different concentrations of CLT. The normalization was carried out with respect to the current transient obtained in the absence of CLT. The composition of the solutions is indicated under "Materials and Methods." i, current; t, time.

 
Considering the absolute Ca2+ requirement for enzyme activation, it is expected that steady state ATPase activity will be inhibited in parallel with inhibition of Ca2+ binding by CLT. In fact, we found that steady state ATPase activity and Ca2+ transport were inhibited by the same CLT concentrations producing inhibition of Ca2+ binding. This was demonstrated by measuring Pi by a colorimetric method after adding ATP to the enzyme in the presence of Ca2+ (Fig. 1) as well as by measuring charge translocation following an ATP concentration jump by the SSM-based technique (Fig. 7). Here again, identical results were obtained by the biochemical and biophysical methods. An interesting observation is that, whereas CLT clearly decreases the amount of moved charge (Fig. 7), the decay kinetics of the remaining current transient is not affected by CLT (not shown). Therefore, although an increasing number of enzyme molecules are inactivated by CLT, the remaining enzyme molecules do not bind CLT and continue to function normally. This suggests a specific inhibitory site for CLT within the ATPase molecule whereby the ATPase molecules are inhibited independent of each other. Furthermore, comparative experiments performed by addition of CLT before or after Ca2+ suggest that the Ca2+-free conformation of the enzyme is more sensitive to CLT and is stabilized by the bound inhibitor.

We also found that phosphoenzyme formation by utilization of ATP in the presence of Ca2+ is inhibited by CLT within the same concentration range as Ca2+ binding (Fig. 2) and ATPase hydrolytic activity (Fig. 1), with a pattern consistent with noncompetitive inhibition. However, phosphoenzyme formation by utilization of Pi in the reverse direction of cycle is only minimally affected by CLT (Fig. 2), as also observed by Snajdrova et al. (5). This reverse reaction is not dependent on activation by Ca2+ and in fact requires removal of bound Ca2+ (36). Therefore, it is apparent that the ATPase catalytic mechanism is not primarily affected by CLT but only secondarily, through interference with activation by Ca2+ binding. Such a specific interference with a partial reaction (i.e. Ca2+ binding) is of interest when compared with the global inhibition of the ATPase cycle produced by TG. So far, 1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene is the only other ATPase inhibitor producing specific inhibition of a single partial reaction, i.e. the E1-P {leftrightarrow} E2-P transition (37).

It should be noted that CLT shows a rather high affinity for SERCA with a KI of 35 µM in the presence of Ca2+ and KI of 7 µM in the absence of Ca2+. Considering the hydrophobic character of the CLT molecule and the location of cation-binding sites in the membrane-bound region of transport ATPases, it is apparent that CLT interferes at this location with the binding of the activating cation and with the conformational change involved in cation binding. In addition to the significance of the CLT effect with regard to the catalytic and possibly pharmacological mechanism, we consider that stabilization of the Ca2+-free, E2 conformation of the enzyme, leaving the catalytic site in a functional state as revealed by the Pi reaction, may be an extremely useful tool in structural and crystallization studies of cation transport ATPases. In fact, ATPase crystal structures of the highest resolution are presently obtained by incorporation of two (rather than one) inhibitors, permitting detection of ordered water molecules and advanced electrostatic calculations yielding estimates of the proton occupancy of acidic residues (38). At the same time, the availability of several inhibitors supports organic synthesis of new, highly specific and potent compounds, guided by the available crystal structure of the ATPase (39, 40).


   FOOTNOTES
 
* This work was supported by the Ente Cassa di Risparmio di Firenze (PROMELAB project) the Ministero dell'Istruzione, dell'Università e della Ricerca, and National Institutes of Health Grant RO1 HL69830. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Florence, Italy. Tel.: 39-055-4573097; Fax: 39-055-4573098; E-mail: guidelli{at}unifi.it.

2 The abbreviations used are: CLT, clotrimazole; SR, sarcoplasmic reticulum; TG, thapsigargin; MOPS, 4-morpholinepropanesulfonic acid; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; MES, 4-morpholineethanesulfonic acid; SSM, solid-supported membrane. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Georgopapadakou, N. H. (1998) Curr. Opin. Microbiol. 1, 547-557[CrossRef][Medline] [Order article via Infotrieve]
  2. Groll, A. H., De Lucca, A. J., and Walsh, T. J. (1998) Trends Microbiol. 6, 117-124[CrossRef][Medline] [Order article via Infotrieve]
  3. Benzaquen, L. R., Brugnara, C., Byers, H. R., Gattoni-Celli, S., and Halperin, J. A. (1995) Nat. Med. 1, 534-540[CrossRef][Medline] [Order article via Infotrieve]
  4. Jan, C. R., Tseng, C. J., Chou, K. J., and Chiang, H. T. (2000) Life Sci. 66, 2289-2296[CrossRef][Medline] [Order article via Infotrieve]
  5. Snajdrova, L., Xu, A., and Narayanan, N. (1998) J. Biol. Chem. 273, 28032-28039[Abstract/Free Full Text]
  6. Andersen, J. P., and Vilsen, B. (1995) FEBS Lett. 359, 101-106[CrossRef][Medline] [Order article via Infotrieve]
  7. MacLennan, D. H., Rice, W. J., and Green, N. M. (1997) J. Biol. Chem. 272, 28815-28818[Free Full Text]
  8. Inesi, G., and Toyoshima, C. (2004) in Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology (Futai, M., Wada, Y., and Kaplan, J. H., eds) pp. 63-87, Wiley-VHC, Weinheim, Germany
  9. Møller, J. V., Nissen, P., Sørensen, T. L., and Maire, M. (2005) Curr. Opin. Struct. Biol. 15, 387-393[CrossRef][Medline] [Order article via Infotrieve]
  10. MacLennan, D. H. (2000) Eur. J. Biochem. 267, 5291-5297[Medline] [Order article via Infotrieve]
  11. Gommans, I. M. P., Vlak, M. H. M., de Haan, A., and van Engelen, B. G. M. (2002) J. Muscle Res. Cell Mot. 23, 59-63[CrossRef][Medline] [Order article via Infotrieve]
  12. Rao, R., and Inesi, G. (2004) in Membrane Transporter Diseases: Molecular Basis of Inherited Transport Defects (Broer, S., and Wagner, C. A., eds) pp. 365-381, Kluwer Academic/Plenum Publishers, New York
  13. Sagara, Y., and Inesi, G. (1991) J. Biol. Chem. 266, 13503-13506[Abstract/Free Full Text]
  14. Seidler, N. W., Jona, I., Vegh, M., and Martonosi, A. (1989) J. Biol. Chem. 264, 17816-17823[Abstract/Free Full Text]
  15. Logan-Smith, M. J., Lockyer, P. J., East, J. M., and Lee A. G. (2001) J. Biol. Chem. 276, 46905-46911[Abstract/Free Full Text]
  16. Sumbilla, C., Lewis, D., Hammerschmidt, T., and Inesi, G. (2002) J. Biol. Chem. 277, 13900-13906[Abstract/Free Full Text]
  17. Khan, Y. M., Wictome, M., East, J. M., and Lee, A. G. (1995) Biochemistry 34, 14385-14393[CrossRef][Medline] [Order article via Infotrieve]
  18. Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566[CrossRef][Medline] [Order article via Infotrieve]
  19. Pintschovius, J., and Fendler, K. (1999) Biophys. J. 76, 814-826[Medline] [Order article via Infotrieve]
  20. Pintschovius, J., Bamberg, E., and Fendler, K. (1999) Biophys. J. 76, 827-836[Medline] [Order article via Infotrieve]
  21. Tadini-Buoninsegni, F., Nassi, P., Nediani, C., Dolfi, A., and Guidelli, R. (2003) Biochim. Biophys. Acta 1611, 70-80[Medline] [Order article via Infotrieve]
  22. Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613[CrossRef][Medline] [Order article via Infotrieve]
  23. Zhou, A., Wozniak, A., Meyer-Lipp, K., Nietschke, M., Jung, H., and Fendler, K. (2004) J. Mol. Biol. 343, 931-942[CrossRef][Medline] [Order article via Infotrieve]
  24. Bartolommei, G., Tadini-Buoninsegni, F., and Moncelli, M. R. (2004) Bioelectrochemistry 63, 157-160[CrossRef][Medline] [Order article via Infotrieve]
  25. Tadini-Buoninsegni, F., Bartolommei, G., Moncelli, M. R., Inesi, G., and Guidelli, R. (2004) Biophys. J. 86, 3671-3686[CrossRef][Medline] [Order article via Infotrieve]
  26. Bamberg, E., Apell, H. J., Dencher, N. A., Sperling, W., Stieve, H., and Läuger, P. (1979) Biophys. Struct. Mech. 5, 277-292[CrossRef]
  27. Eletr, S., and Inesi, G. (1972) Biochim. Biophys. Acta 282, 174-179[Medline] [Order article via Infotrieve]
  28. Patton, C., Thompson, S., and Epel, D. (2004) Cell Calcium 35, 427-431[CrossRef][Medline] [Order article via Infotrieve]
  29. Hartung, K., Froehlich, J. P., and Fendler, K. (1997) Biophys. J. 72, 2503-2514[Medline] [Order article via Infotrieve]
  30. Lanzetta, P. A., Alvarez, L. J., Reinach, P. S., and Candia, O. A. (1979) Anal. Biochem. 100, 95-97[CrossRef][Medline] [Order article via Infotrieve]
  31. Seifert, K., Fendler, K., and Bamberg, E. (1993) Biophys. J. 64, 384-391[Medline] [Order article via Infotrieve]
  32. Läuger, P. (1991) Electrogenic Ion Pumps, pp. 104-136, Sinauer Associates, Sunderland, MA
  33. Inesi, G., Kurzmack, M., Coan, C., and Lewis, D. E. (1980) J. Biol. Chem. 255, 3025-3031[Free Full Text]
  34. Lax, A., Soler, F., and Fernandez-Belda, F. (2002) Am. J. Physiol. 283, C85-C92
  35. Butscher, C., Roudna, M., and Apell, H. J. (1999) J. Membr. Biol. 168, 169-181[CrossRef][Medline] [Order article via Infotrieve]
  36. Masuda, H., and de Meis, L. (1973) Biochemistry 12, 4581-4585[CrossRef][Medline] [Order article via Infotrieve]
  37. Hua, S., Xu, C., Ma, H., and Inesi, G. (2005) J. Biol. Chem. 280, 17579-17583[Abstract/Free Full Text]
  38. Obara, K., Miyashita, N., Xu, C., Toyoshima, I., Sugita, Y., and Toyoshima, C. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 14489-14496[Abstract/Free Full Text]
  39. Uhlemann, A. C., Cameron, A., Eckstein-Ludwig, U., Fischbarg, J., Iserovich, P., Zuniga, F. A., East, M., Lee, A., Brady, L., Haynes, R. K., and Krishna, S. (2005) Nat. Struct. Mol. Biol. 12, 628-629[CrossRef][Medline] [Order article via Infotrieve]
  40. Sohoel, H., Liljefors, T., Ley, S. V., Oliver, S. F., Antonello, A., Smith, M. D., Olsen, C. E., Isaacs, J. T., and Christensen, S. B. (2005) J. Med. Chem. 48, 7005-7011[CrossRef][Medline] [Order article via Infotrieve]

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