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Department of Biology, University of Konstanz, Konstanz, Germany
Correspondence: Address reprint requests to Hans-Jürgen Apell, Dept. of Biology, University of Konstanz, Fach M635, 78457 Konstanz, Germany. Tel.: 49-7531-88-2253; Fax: 49-7531-3183; E-mail: h-j.apell{at}uni-konstanz.de.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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) E1 CaE1 Ca2E1 can be characterized by two time constants, 
1 and 2, both of which are not significantly Ca2+-concentration-dependent and only weakly pH-dependent at pH < 7.5. Both time constants differ by a factor of 
50 (4.7 vs. 200 ms). The weak substrate-dependence indicates that the rate-limiting process is not related to Ca2+ migration through the access channel and ion binding to the binding sites but to conformational rearrangements preceding the ion movements. The high activation energy obtained for both processes, 42.3 kJ mol–1 and 60.3 kJ mol–1 at pH 7.2, support this concept. Transient binding of Ca ions to the loop L67 and a movement of the Ca-loaded loop are discussed as a mechanism that facilitates the entrance of both Ca ions into the access channel to the ion-binding sites. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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). Performing this task the enzyme undergoes different states while running through its pump cycle. This cycle, the so-called Post-Albers cycle, describes transport of two Ca2+ from the cytosol to the SR lumen and, subsequently, counters transport of two H+ in a Ping-Pong mode while the enzyme is phosphorylated in the first and dephosphorylated in the second half cycle. The enzymatic partial reactions catalyze the transition between both basic conformations, E1, in which the ion-binding sites are accessible from the cytoplasm, and P-E2, in which the binding sites are opened to the luminal aqueous phase. It is shown that the ion-binding and release steps in both conformations are electrogenic, i.e., net electric charges are moved into and out of the membrane dielectric (2,3). These findings can be explained by the concept that the ion-binding sites are located (almost) in the middle of the membrane domain of the SR Ca-ATPase (4). Recently published structures of the SR Ca-ATPase in four different states, Ca2E1, ATP·E1 (stabilized by E1·AMPPCP), PE2, (obtained as 
), and E2·Thapsigargin confirm this claim (5–10). Although these structures present snapshots of defined enzyme conformations, kinetical investigations provide information about conformational rearrangements that the pumps have to go through when they convert from one state to another.
Since binding (or release) of ions to (or from) the SR Ca-ATPase is electrogenic, these reaction steps can be detected by the styryl dye 2BITC (11). Binding and release of H+ and Ca2+ in both states have been analyzed by equilibrium titration experiments, and the respective dissociation constants were obtained (2).
To determine the rate constants that control the transition between different enzyme states, caged compounds were used to produce concentration jumps of pump substrates to trigger transitions into new equilibrium states. After a flash-induced release of ATP from its inactive precursor, caged ATP, in the presence of cytoplasmic Ca2+, the Ca-ATPase undergoes enzyme phosphorylation, Ca2E1 
(Ca2)E1-P, conformation transition, (Ca2)E1-P P-E2Ca2, Ca2+ release, P-E2Ca2 P-E2, and binding of protons, P-E2 P-E2H2. Such experiments were used to determine the rate-limiting steps of the partial reactions, the conformation transition, and luminal H+ binding (3). Another substrate of the Ca-ATPase is the transported ion, Ca2+. In 1988, the application of a caged Ca was introduced to characterize Ca2+ binding to the enzyme (12,13). The cage, DM-Nitrophen, is a photolabile chelator of Ca2+ that is split by an intense ultraviolet (UV) light flash. This compound is commercially available, and allows a release of Ca2+ within less than a microsecond.
Since the reaction HE1 Ca2E2 is electrogenic, it can be detected by the fluorescent electrochromic styryl dye 2BITC (2), and due to a response time of the dye far below a microsecond, charge movements as fast as the Ca2+ release from its caged chelator can be detected. This reaction sequence includes not only the release of protons from the pump and Ca2+ binding to the pump but also conformational rearrangements of the protein, which were detected previously by spectroscopic methods (14,15). Experiments with radioactive Ca2+ have shown that the two Ca2+ are bound to the E1 conformation in an ordered and sequential fashion (16–19). Binding of Ca2+ is cooperative (17), binding of the first Ca2+ induces (formation and) access to a binding site for the second Ca2+ (20). After binding of the second Ca2+ enzyme activation occurs (21). The presence of two defined Ca2+ binding sites, I and II, led to the model of Ca2+ single-file binding (22). The entry path of Ca2+ from the cytosol to the binding sites I and II is yet not fully identified, although there is evidence that it is lined up with polar residues (23). There is no water-filled vestibule visible as it is observed in ion channels (24).
According to a recent review, Glu309 may play a role in gating the ions to the binding sites (23). This model was also proposed from mutagenesis studies (25). After the first Ca2+ has bound, the second Ca2+ is binding to site II with a higher affinity, since the region of Asp800 looses flexibility after binding of the first Ca2+ (26).
In this study, we present time-resolved experiments of Ca2+ binding to the SR Ca-ATPase. The dependence of the rate constants on Ca2+ concentration, pH, and temperature support the concept of a conformational relaxation between binding of the first and second Ca2+, and allowed us to introduce constraints for possible mechanisms of structure-function models.
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MATERIALS AND METHODS |
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) was a gift from Dr. H.-D. Martin, University of Düsseldorf, Düsseldorf, Germany. All other reagents were the highest grade commercially available.
Enzyme preparations and reconstitution
Ca-ATPase was prepared by a slight modification of the method of Heilmann et al. (28) from the psoas muscles of rabbits. The whole procedure was performed at temperatures below 4°C. The determination of the protein content of the membrane preparation was performed according to Markwell. The most active fractions of the final density gradient separation had a protein content of 2–3 mg ml–1. The enzyme activity was determined by the linked pyruvate kinase/lactate dehydrogenase assay. Background enzyme activity of the isolated preparation was measured by addition of 1 µM thapsigargin that blocks the SR Ca-ATPase completely. The Ca-ATPase-specific activity was 175 µmol Pi per mg protein and h at 37°C and could be increased up to 310 µmol Pi per mg protein and h in the presence of A23187 to short-circuit the vesicles forming membranes for Ca2+. With a molecular weight of 110,000 g mol–1 and a specific activity of 5.2 units mg–1, the turnover rate of the pump is 9.5 s–1 in this preparation. In control experiments, the effect of the styryl dye 2BITC on the enzymatic activity was checked. Up to a dye concentration of 1.2 mM, no changes of the enzymatic activity could be observed.
Detection of partial reactions with 2BITC
The fluorescence measurements in equilibrium-titration experiments were performed with a self-constructed setup using a HeNe laser with a wavelength of 543 nm (Laser 2000, Wessling, Germany) to excite the fluorescence of the electrochromic dye 2BITC (2). The emitted light was collected perpendicularly to the incident light, filtered by a narrow-band interference filter (
max = 589 nm, half-width 10.6 nm) and detected by a head-on photo multiplier (R2066, Hamamatsu Photonics, Hamamatsu City, Japan). The photo current was amplified by a Keithley current amplifier 427 (Keithley Instruments, Cleveland, OH) and collected by a data-acquisition board of a PC (PCI-T112, Imtec, Backnang, Germany) with sampling frequencies between 1 and 10 Hz. The experimental data were displayed on the monitor, stored and analyzed on the PC. The temperature in the cuvette (2 ml) was maintained by a thermostat at 20°C.
For data recording of fluorescence signals with high time-resolution a setup was used, whose design was published earlier (3). A cylindrical quartz cuvette (internal diameter, 7.8 mm) containing 300 µl buffer (layer height, 5 mm) was placed in the upper focus of an ellipsoidal mirror (Melles-Griot, Zevenaar, Netherlands) whose opening was directed downwards. The buffer contained 600 nM 2BITC, 18 µg Ca-ATPase preparation, 50 µM DM-Nitrophen and the pH was adjusted by HCl to certain values. The residual concentration of Ca2+ was estimated to 14 µM. The dye was excited by a 543-nm HeNe laser from the top of the setup. A quartz lens was adjusted to widen the laser beam and to illuminate the whole solution almost homogeneously. The emitted light was collected by the ellipsoidal mirror and reflected into the second focus of the mirror. An interference light filter (589 ± 10 nm) selected the emitted light of the styryl dye before passing the entrance window of a photo multiplier (R928, Hamamatsu Photonics). The output current was amplified by an I/V converter and fed into a 12-bit data-acquisition board of a PC with sampling frequencies between 1 and 500 kHz. The bottom of the cuvette was in contact with a thermostated copper socket (that also stopped the incident light). To release Ca2+ from its precursor, caged Ca, an UV-light flash (wavelength 350 nm, maximum power 6 MW) was generated by an EMG 100 excimer laser (Lambda Physics, Göttingen, Germany) and directed through a quartz lens into the cuvette, illuminating the whole buffer volume. The amount of released Ca2+ was dependent on the intensity of the UV flash and buffer pH. Approximately 30 nM at high pH and 100 nM at low pH could be obtained by the first flash. By successive light flashes, the Ca2+ concentration could be increased stepwise. Typically, four flashes had to be applied to saturate the binding sites of the SR Ca-ATPase with Ca2+. Further flash-induced Ca2+ release induced no further fluorescence changes. To calibrate the respective Ca2+ concentrations after a flash-induced concentration jump, equilibrium titration experiments were performed in corresponding buffers at various pH, and the relative fluorescence changes and the known Ca2+ concentrations could be correlated with high precision (2).
To optimize the signal/noise ratio, seven measurements under identical conditions were averaged. The time course of the fluorescence signal was analyzed by a numerical fit of the data with a sum of two exponential functions,
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1 and 2, as well as the respective fluorescence amplitudes, F1 and F2, and the new stationary fluorescence level, F
, after the relaxation process, which was used to determine the Ca2+ concentration. |
RESULTS |
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3 nM Ca2+. The Ca2+ concentration was increased in a fast, steplike manner by a UV flash to 30 nM, as could be determined from the fluorescence decrease. Subsequent flashes led to further decreases of the fluorescence amplitude until, at a concentration of 200 nM (at pH 8.2), a saturation of the binding sites was reached (Fig. 1 A).
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1 of the faster and 2 of the slower process. The corresponding fluorescence amplitudes account for the electrogenic Ca2+ binding to the binding sites of the enzyme. These fluorescence amplitudes do not necessarily represent the rate-limiting step if the electrogenic ion movements inside the membrane domain are separate and fast (i.e., diffusion-controlled) processes. The time constant of the fast process, 1, ranged from 2 to 8 ms and that of the slow process, 2, from 50 to 200 ms. A successful approach to identify the molecular processes controlling the time course of the fluorescence changes is the investigation of their dependence on the Ca2+ concentration and pH. Ca2+ and H+ are able to enter the ion-binding sites, and may interact additionally with other moieties of the protein surface, thus producing allosteric effects, especially in the case of protons. Therefore, detailed studies on the dependency of the kinetics on both substrates, Ca2+ and H+, and on the temperature, were performed.
Ca2+ concentration dependence
Since the observed fluorescence changes upon Ca2+ release were always decreasing signals, it was clear that, according to the detection mechanism of the styryl dye 2BITC, a (net) uptake of positive charges, i.e., Ca2+, into the membrane domain of the pumps were observed (11). Therefore, it could be expected that the kinetics of this process would be affected by the free Ca2+ concentration. The concentration range in which experiments could be analyzed was between 18 nM and 2 µM. Fig. 2 A shows the result of the analysis of the time-dependent fluorescence decrease after the flash-induced Ca2+-concentration jump. As illustrated in Fig. 1 B, the time course could be fitted and characterized by two time constants, 1 and 2. In the range above 50 nM, both processes were not significantly dependent on the Ca2+ concentration. The fast process could be described by an average time constant, 1, of 4.7 ± 0.5 ms (without the data points <50 nM). This corresponds to a rate constant of 213 s–1. The slow process had an average time constant, 2, of 0.20 ± 0.01 s, or a respective rate constant of 5 s–1. The deviating smaller time constants at low Ca2+ concentrations (<50 nM) could be obtained only in buffer with high pH, due to the pH-dependent binding affinity of caged Ca. Therefore, the lowest data points do not show a real Ca2+ dependence but a pH effect (see below). The ratio of both time constants was approximately a factor of 50, and both processes controlling the rate-limiting steps are obviously not reactions that are dependent on the free Ca2+ concentration in the buffer.
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). The maximum value of F1 was –2% ± 0.3%. The amplitude F2 was not significantly dependent on the Ca2+ concentration and had a value of –2.7% ± 0.3%.
pH dependence
Corresponding to the experiments shown in Fig. 1, measurements were performed in buffers at various pH values in the range between 6.3 and 8.2. In the whole pH range, the fluorescence decrease showed a time course that had to be fitted by two exponentials. Both the fast and the slow processes were, in a similar manner, dependent on pH, as shown in Fig. 3. Independent of the pH, the ratio between both time constants was again 50. The lines in Fig. 3 are drawn to guide the eye; they have the same shape, and are shifted only by a factor of 50. The larger error bars of 1 were caused by the smaller amplitude of the fast process (20% of the total amplitude). At higher proton concentrations in the buffer, both processes were slowed down.
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1 and 2, were determined in a temperature range from 7°C to 36°C. The experiments were performed with initially negligible Ca2+ concentrations (<3 nM), and in buffers with pH of 6.6, 7.2, and 7.8. To analyze the temperature dependence, the time constants were represented as an Arrhenius plot, as shown in Fig. 4. The data sets obtained from measurements in the same buffer could be fitted by a regression line. The slope in the semilogarithmic plot is proportional to the activation energy of the rate-limiting process. For the temperature dependence of both time constants, different activation energies were found. The fast process showed an activation energy of 31.6 kJ mol–1 at pH 7.8, 42.3 kJ mol–1 at pH 7.2, and 50.6 kJ mol–1 at pH 6.6. The activation energy of the fast process is higher at lower pH. The slow process was characterized by an activation energy of 72.3 kJ mol–1 at pH 7.8, 65.2 kJ mol–1 at pH 7.2, and 60.3 kJ mol–1 at pH 6.6. The activation energy of the fast process is higher at higher pH.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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). This method was applied to investigate Ca2+ binding to the SR Ca-ATPase for the first time in 1990 (30). In this study, the time dependence of the extravesicular Ca2+ concentration was monitored, and upon release from its cage, the Ca2+ concentration increased and, subsequently, due to the action of the Ca pump, a reduction of the free Ca2+ was observed. The time resolution was in the order of 20 ms. In the experiments presented here, Ca2+ uptake into the membrane domain of the Ca-ATPase was detected with a time resolution of typically 100 µs.
After the UV flash-triggered release of Ca2+ resulting in concentration jumps between 30 nM and 450 nM, two processes could be resolved by analysis of the 2BITC-fluorescence decrease with Eq. 1. The fast process is described by 1, which had values between 2 ms and 8 ms, and the slow process with 50 ms < 2 < 200 ms. We estimated the dependence of the two processes on pH, Ca2+ concentration and temperature.
Both time constants were not significantly dependent on the free Ca2+ concentration above the limiting 50 nM. The time constant of the faster process, 1, was averaged to 4.7 ± 0.5 ms at T = 20°C. A small increase of 1 could be observed in the range of pH < 7.5 (Fig. 3). This tendency became more evident when the temperature dependence of 1 was investigated at different pH values, as shown in Fig. 4. The Arrhenius plot reveals a clear shift of the values of 1/1 in buffer of pH 7.8 when compared to pH 7.2 and pH 6.6. And, what is more significant, the activation energy of the fast process, as determined from the temperature-dependence of 1/1, was pH-dependent too—namely, 31.6 kJ mol–1 at pH 7.8, 42.3 kJ mol–1 at pH 7.2, and 50.6 kJ mol–1 at pH 6.6. The activation energy of the fast process was increased at higher concentrations of protons.
Similarly to the faster process, the time-constant of the slower process, 2, was also independent of the Ca2+ concentration and approximately a factor-of-50 larger than 1 (Fig. 2 A). It is, in the same way, pH-dependent as 1 (Fig. 2 A). When the pH was reduced from 8.15 to 6.28, 2 increases by a factor of 3.5. Significant differences were also found in the analysis of the temperature dependence. The Arrhenius plot showed a higher activation energy for the slower process than for the fast one at all buffer pH values. The activation energy for the slower process decreased with the proton concentration of the buffer and is 60.3 kJ mol–1 at pH 6.6, 65.2 kJ mol–1 at pH 7.2, and 72.3 kJ mol–1 at pH 7.8.
The reaction steps that follow the flash-induced Ca2+ release occur according to the Post-Albers scheme with the sequence HxE1 E1 CayE1, with 0 
x 3 and 0 y 2 (x is the number of protons bound to the ion pump in the E1 conformation and depends on buffer pH; y is the number of Ca2+ bound after the concentration jump, when the protein reached equilibrium). The number of bound protons in E1 state is discussed in literature to be two, three, or four (31–34). The binding of protons is characterized to be in competition with the binding of other ion species (35). We were able to simulate the binding of protons to the E1 state with a linear binding scheme and, according to Peinelt and Apell (2), the distribution of the different states are at pH 8 30% E1, 30% HE1, 30% H2E1, and at pH 6 50% H2E1, 50% H3E1.
In contrast to the time constant 1, the respective fluorescence amplitude F1 showed a binding affinity for Ca2+ in agreement with the published equilibrium dissociation constants for Ca2+. This observation may be explained by the fact that the rate constant is controlled by the rate-limiting step of the relaxation process, which is not necessarily the Ca2+ migration into the binding site and binding inside the membrane domain of the protein, whereas the fluorescence decrease reports the amount of positive charge in the binding sites. The apparently Ca2+-independent fluorescence amplitude of the slower process, F2, has to be seen in the light of the high binding affinity of the second Ca2+ site. Therefore, it can be expected that the second Ca2+ is immediately bound when the site becomes available at the given Ca2+ concentrations (2). The maximum fluorescence decrease of –2% for the fast process and –2.7% for the slow process indicate that less (net) charge was imported during the faster first reaction step. This may be explained by the fact that due to a pK 8 of the binding sites in E1 (2), part of the binding sites were occupied by a proton which is exchanged against a Ca2+, thus reducing the net amount of charge imported.
The analyzed processes cannot be explained as simple binding of ions. First, ion binding would be a second-order reaction and, therefore, the rate constant has to be Ca2+-concentration dependent. Second, ion binding is a diffusion-controlled process, which would occur in a time period below 1 ms. Third, simple binding of ions cannot account for the observed high activation energy. Ion binding in terms of a transfer from its hydrated form into a moiety mimicking a hydration shell has typically a low activation energy in the order of 5–10 kJ mol–1.
Ca2+ binding to the E1 conformation from the cytosol was shown to be a single-file process by Ca2+ exchange experiments performed with radioactive Ca2+ by Inesi and collaborators (1,17,22). The first ion binds and causes a conformation relaxation, a pre-occlusion of the first ion, and the high-affinity binding site for the second ion then becomes accessible (17,36). A similar conformational rearrangement may be postulated also for the enzyme after the release of the (last) proton and before binding of the first Ca2+. Therefore, the linear ion-binding and release sequence of the Post-Albers cycle in the E1 conformation is expanded by two additional steps whose kinetics are slow in comparison to the electrogenic diffusion of the ions and their coordination in the sites:
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1 and 2, are interpreted as relaxation times of the reaction steps, 
and 
respectively, and are functions of the forward and backward rate constants, = (kfor + kback)–1. The rate constants themselves could not be deduced with the experiments performed. A comprehensive reaction model has to account for the fact that the kinetics is independent of the free Ca2+ concentration in the buffer and only weakly dependent on pH. Each of the two processes facilitates binding of one Ca2+, and the faster process displaces, in addition, one proton from inside the membrane domain. Both processes should include conformational relaxations to account for the high activation energy.
With respect to the structure of the membrane domain of the SR Ca-ATPase, the membrane helix M6 and the loop L67 are promising candidates to suggest a molecular mechanism that explains the observed behavior. In the literature, pre-ion-binding of Ca2+ to the cytoplasmic loop L67 is already introduced. It is discussed as a transfer location for the ions into their binding sites inside the membrane domain (37,38). This loop is located between the sixth and seventh transmembrane helix and consists of a 20-amino-acid peptide beginning with Gly808 close to the entrance to the ion-binding sites. A synthetic peptide of identical sequence was shown to form Ca2+ complexes (39), and Ca-ATPase mutants with two or three of the aspartate residues in the L67 sequence replaced by alanine reveal a significantly lower Ca-ATPase activity than the wild-type (37). Lenoir and collaborators showed by fluorescence experiments with isothiocyanate-labeled protein a reduced Ca2+ affinity in Ca-ATPase mutants modified at Asp813, Asp815, and Asp818 (40). Loop L67 may act as a guidance device by transiently binding a Ca2+ to these three aspartate side chains and facilitating the entry mechanism of the ions (38), probably by a movement of the ion-loaded loop.
Although Lenoir and collaborators modified their proposal on the role of the L67 loop (40), such a L67-supported Ca2+-entry mechanism would provide a suitable explanation for the experimental findings presented here. The two analyzed processes are assumed to be a movement of L67 as rate-limiting steps before binding of each Ca2+ inside the enzyme, and can be identified as the two conformational relaxations, whereby the time-constant 1 represents 
and 2 represents 
Assuming that loading of the ion-binding sites is more effective when facilitated by a L67-loop movement, the first bound Ca2+ will reduce the mobility of L67 by coordination with Thr799 and Asp800 on the helix M6. On one hand such a fixation of M6 by the Ca2+ might be essential for the formation of the second Ca2+-binding site, and on the other hand it may explain also the deceleration of the kinetics of the second partial reaction analyzed, 
This concept is supported by the significantly lower activation energies of 30–50 kJ mol–1 for the fast process compared to 60–72 kJ mol–1 for the slow process. Binding of the first Ca2+ inside the protein affects M6, and therefore, it can change the kinetical properties of the conformational relaxation of L67 loaded with the second Ca2+. The activation energy of the L67 movement loaded with the second Ca2+ will be higher than the L67 movement with the first Ca2+ ion and a non-occupied binding site I. The comparable pH dependence of both time constants cannot be assigned to interactions of the protons with the high-affinity Ca2+ binding sites of the pump due to the fact that no significant competition with Ca2+ was observed. An alternative explanation could be the binding of a proton to an allosteric site from which it has to dissociate before the conformational relaxation may occur. A possible mechanistic explanation can also be related to loop L67, which contains three aspartate residues (Asp813, Asp815, and Asp818) whose carboxylic side chains might be protonated. A buffer pH below 7 could result in a protonation of one (or more) aspartate residues and thereby slightly reduce the Ca2+ affinity. If only the Ca-loaded-L67 is able to perform a conformational rearrangement, the rate of 2BITC-detected Ca2+ binding to site I will be decreased at low pH. The slower Ca2+ binding at the L67 site leads to increased 1 and 2 at low pH as shown in Fig. 3.
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CONCLUSION |
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1 and 2, which precede the electrogenic binding of the first and the second Ca2+. This result is in agreement with the observation of published binding studies with tracer Ca2+, showing that the second binding site becomes available only after occupation of the first site. The data also support the proposal that each Ca2+ is bound transiently to the cytoplasmic loop L67, which provides an entry mechanism for Ca2+ by a spatial rearrangement to guide the ions into the access channel to the binding sites inside the membrane domain of the pump. |
ACKNOWLEDGEMENTS |
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This work was financially supported by the Deutsche Forschungsgemeinschaft (Ap 45/4).
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FOOTNOTES |
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Submitted on June 9, 2005; accepted for publication July 13, 2005.
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REFERENCES |
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