The activity of the SR ()calcium release channel is controlled by the cytoplasmic Ca concentration in a biphasic fashion, as shown in efflux studies with SR vesicles (1, 2, 3) and channel conductance measurements of purified JFP incorporated into bilayers(4) . Thus, the channel is activated at lower [Ca] with AC 0.5 µM and inhibited at higher [Ca] with IC 0.15 mM, suggesting that there are two classes of calcium sites involved in the channel regulation. An abrupt increase of [Ca] in the activating range of [Ca] induces SR calcium release (5) as well as transient increase in channel open probability(6) .

In the case of the voltage-dependent Ca transient in the skeletal muscle fiber system, however, the modes of regulation of SR calcium release by the cytoplasmic Ca are rather complex as seen in the considerable controversy existing in the literature(7, 8, 9) . According to one view, an abrupt increase in the concentration of the sarcoplasmic free Ca (a Ca jump) resulting from the depolarization-induced SR calcium release leads to a rapid attenuation of release flux(10, 11, 12, 13, 14) . According to the widely referred model originally proposed by Schneider and Simon(12) , rapid binding of the released calcium to the JFP is followed by a slow conformational change, leading to an inactivation of the channel. According to the opposing view, the Ca jump activates SR calcium release probably by a Ca-induced Ca release mechanism(15, 16, 17) . Such a positive feedback mechanism would be important for the activation of the group of JFPs which is not physically coupled with the T-tubule voltage sensor(18) . A considerable amount of evidence also suggests that the binding of the released Ca to the T-tubule voltage sensor re-activates the group of JFPs that has been stimulated by mediation of the voltage sensor(19, 20, 21, 22, 23, 24, 25) . The most frequently used method in the literature to distinguish Ca-induced inactivation versus Ca-induced activation of calcium release is to try to suppress the changes in the cytoplasmic [Ca] with rapidly reacting high affinity calcium buffers such as BAPTA or fura-2. In this test, a decrease of the release rate suggests Ca activation, while an increase suggests Ca attenuation. Such experiments have been carried out by several laboratories with controversial results. For example, the intracellular fura-2 (2-3 mM) activated the release rate in both voltage-clamped and action potential-stimulated fibers, in favor of the view of Ca inactivation(26, 27, 28) . Conversely, an equivalent concentration of BAPTA or fura-2 virtually eliminated depolarization-induced Ca transient under voltage-clamped conditions in favor of Ca activation(15) . A similar blocking effect by 4 mM BAPTA was also reported in E-C coupling in cell homogenates of rabbit skeletal muscle(29) .

The isolated triad system, which mimics physiological E-C coupling (30) and is freed from cytoplasmic calcium-binding proteins, would permit straightforward analysis of the calcium release time course. The main purpose of this study is to investigate how the changes in the concentration of BAPTA-calcium buffer ([Ca] = 0.1 µM) affect the kinetics of the JFP conformational change and SR calcium release induced by T-tubule depolarization, using the isolated triad system. As shown here, there was an alteration in the kinetics of both protein conformational change and calcium release when the concentration of BAPTA was varied. Thus, stimulation of the JFP at lower [BAPTA] produced biphasic protein conformational changes, as evidenced by a biphasic increase of the fluorescence intensity of the JFP-bound probe, MCA. The second phase, but not the first phase, was reduced upon increasing the BAPTA concentration. Conversely, the Ca jump, applied concurrently with T-tubule depolarization, from 0.1 µM up to 1.0 µM increased the magnitude of the second phase of protein conformational change without affecting the first phase, suggesting that the second phase of protein conformational change represents a secondary activation of JFP by the released Ca. At lower concentrations of BAPTA, under conditions in which an increase in the [Ca] in the vicinity of the Ca channel (Ca) was sufficiently large, calcium release was accelerated in an early phase and rapidly reached a maximal level. Analysis of the d(calcium release)/dt curves suggested that both initial increase and subsequent decrease in the calcium release rate by Ca occurred in a coupled manner. Thus, it appears that calcium release involves two sequential reaction cycles as follows. In the first cycle, the triggering signal applied to the JFP moiety of the triad via the T-tubule produces conformational changes in the JFP to open the calcium release channel and to induce calcium release from the SR. The released Ca stimulates the JFP again, inducing the second cycle of the reaction involving protein conformational change SR calcium release.

The present results suggest that the released Ca accelerates both initial activation and subsequent attenuation of calcium release in a synchronized fashion. This mechanism provides a reasonable explanation for both of the two controversial views in the literature described above.

EXPERIMENTAL PROCEDURES

Preparations

Site-specific Fluorescent Labeling of the JFP Moiety of the Triad

Assays of Protein Conformational Changes in the JFP Moiety of the Triad Induced by T-tubule Depolarization and Subsequent Calcium Release from SR

Priming Solution

Depolarization Solution

Control Solution

The time courses of SR calcium release were monitored with a stopped-flow fluorometer (BioLogic SFM-3 with MOS-200 optical system) using fluo-3 as a Ca indicator (excitation at 437 nm, emission at 530 nm with a 510-nm cut-off filter). Approximately 30 traces of the fluo-3 signal were averaged for each experiment.

In some experiments, a Ca jump was applied concomitantly with T-tubule depolarization or in non-depolarizing conditions. For this purpose, the triads were incubated in the priming solution for 6-7 min, and [Ca] was adjusted to 0.1 µM by adding 10 mM BAPTA-calcium buffer. Then, 15 µl of solution of the primed triads was mixed with 135 µl of depolarizing solution or non-depolarizing control solution containing 10 mM BAPTA-calcium buffer adjusted to various concentrations of Ca in a range of 0.1-1.0 µM.

The time courses of fluorescence change of the protein-bound MCA (excitation at 368 nm, emission at 440 nm using an interference filter with 70-nm bandwidth) induced by depolarization were monitored with the same stopped-flow fluorometer. Approximately 60 traces of the MCA fluorescence signal were averaged for each experiment.

To calculate the net calcium release (nmol/mg) from the fluorescence intensity of fluo-3 in the presence of various concentrations of BAPTA-calcium buffer, each of the 1000 data points of the fluo-3 trace was calculated by using appropriate association constants for all ligands and metals present in the reaction solutions. In this calculation, we assumed that the rates of binding of the released calcium to the buffer (35) and to fluo-3 (36) were significantly higher than the rate of calcium release itself. The coefficient of (fluo-3 signal)/(the change in [Ca]), required for the above calculation, was determined at each [Ca] in the range of 0.01-1.0 µM adjusted with the BAPTA-calcium buffer.

RESULTS

Effects of Released Caon the Depolarization-induced JFP Conformational Change

Figure 1: A, top of panel, time courses of the changes in the fluorescence intensity of the MCA probe attached to the JFP moiety of the triad (namely conformational changes in JFP) upon chemical depolarization in the presence of various concentrations of BAPTA at 0.1 µM [Ca]: a, 0.1 mM; b, 0.2 mM; c, 0.4 mM; d, 10 mM. Bottomofpanel, Ca-dependent portion of conformational change calculated by subtraction of the curve obtained at 10 mM BAPTA from those obtained with lower concentration of BAPTA. B, time courses of the MCA fluorescence change lack the second phase when T-tubule was depolarized in the presence of 0.1 mM BAPTA at 0.1 µM [Ca] without loading the SR moiety with calcium: see curvea(-Ca). Curvesa and d are the same experiments as those in A. Each trace was obtained by signal-averaging a total of 180-300 traces originating from three to five different experiments. The curves were traced by fitting a double-exponential model: y = A(1 -e

In the experiment shown in Fig. 1B (curvea(-Ca)), we carried out the same experiment as in Fig. 1A (curvea, 0.1 mM BAPTA), but without calcium loading of the SR moiety. Under these conditions, T-tubule depolarization would induce JFP protein conformational change but no calcium release. As seen, protein conformational change showed a monophasic time course, which was essentially identical to the curve obtained with calcium-loaded triads in the presence of 10 mM BAPTA (Fig. 1B, curved). This is again consistent with the notion that the second phase of protein conformational change is produced by the released Ca.

In order to assess the magnitude of Ca that is required to produce the comparable size of the second phase of protein conformational change observed in Fig. 1A, we generated, concurrently with T-tubule depolarization (Fig. 2A) or dilution with the non-depolarizing control solution (Fig. 2B), various levels of Ca jump from 0.1 µM up to 1.0 µM using strong BAPTA-calcium buffers. As shown in Fig. 2A, the Ca jump together with T-tubule depolarization produced the second phase of protein conformational change, with virtually no effect on the first phase. The Ca jump without depolarization produced a monophasic slow conformational change (Fig. 2B); there was no voltage-dependent rapid conformational change in this case. Interestingly, the curves obtained without depolarization (Fig. 2B) show a surprising similarity to those curves representing the Ca-dependent portion of the depolarization-induced conformational change shown in the lower half of Fig. 1A.

Figure 2: Time courses of the changes in the fluorescence intensity of the MCA probe attached to the JFP moiety of the triad upon application of various levels of Ca jump concomitantly with dilution with the depolarization solution (A) or the non-depolarizing solution (B). The Ca jump was from 0.1 µM to various levels (in µM) as indicated. The MCA-labeled triads were incubated in a priming solution containing loading calcium as described under ``Experimental Procedures,'' and the solution [Ca] was adjusted to 0.1 µM by adding 10 mM BAPTA-calcium buffer. The primed triads were then mixed with the depolarizing (A) or non-depolarizing control (B) solutions buffered at various [Ca]s as indicated. Each trace was obtained by signal-averaging a total of 240 traces originating from four different experiments.

Released CaAccelerates Both Activation and Subsequent Attenuation of Depolarization-induced Calcium Release

Figure 3: Time courses of calcium release (A), d(calcium release)/dt (B), and the changes in [Ca] induced by chemical depolarization in the presence of various concentrations of BAPTA at 0.1 µM [Ca] (C): a, 0.1 mM; b, 0.2 mM; c, 0.4 mM. Inset to panelA, the same data shown in a longer time scale. Each trace was obtained by signal-averaging a total of 90 traces originating from three different experiments. To obtain the calcium flux curves shown in B, first derivatives of the Ca curves (A) were calculated. The resultant d(calcium release)/dt curves were fitted by the following equation, which is the solution of Model 1 describing R,

Figure S1: Scheme 1.

Fig. 3C illustrates the time course of the changes in the free Ca concentration during the depolarization-induced calcium release reaction in the presence of three different concentrations of BAPTA. As seen, the maximum limit of Ca produced in the reaction solution was not more than 30 nM. If we consider the changes of Ca during the earlier phase of the reaction where sharp changes occurred in the d(calcium release)/dt curve, the Ca value affecting the release kinetics would have been in a range of several nM. From the results of the Ca jump experiment shown in Fig. 2, we postulate that the Ca which is actually affecting the release kinetics is not a several nM Ca in the reaction solution (Fig. 3C) but a submicromolar Ca jump (Fig. 2), which presumably is occurring in a compartmentalized region within the JFP (see ``Discussion'').

DISCUSSION

One of the important unsettled questions in muscle physiology is as to whether the transient increase in the sarcoplasmic free Ca concentration due to voltage-dependent activation of skeletal muscle fiber (i.e. Ca) produces positive or negative feedback effects on SR calcium release, as outlined in the ``Introduction.'' In the present study we addressed this question using our in vitro E-C coupling assay system, which offers several advantages as follows. Chemical depolarizationinduced calcium release in this system mimics physiological E-C coupling(30) , so that it provides a simplified model useful for the analysis of physiological E-C coupling at the molecular level. In particular, the purified vesicles are freed from cytoplasmic Ca binding proteins and permit an unambiguous control of the ionic milieu of the reaction solution, e.g. Ca. The system also permits straightforward studies of the calcium release time course.

The most important aspect of this study is that we could deduce several novel features concerning the feedback effects of released Ca on the kinetics of conformational changes in the JFP and SR calcium release induced by T-tubule depolarization. The present studies on the protein conformational change have permitted clear insight into the modes of regulation of Ca channel by the released Ca. The triggering signal applied to the JFP via T-tubule induces biphasic changes in its protein conformation. Only the second phase of protein conformational change seems to represent the portion of conformational change that is induced by the Ca released from SR, because as shown here the effects of varying the BAPTA concentration are manifested primarily on the second phase, and the secondary conformational change occurs usually after an appreciable latent period. Furthermore, increasing the levels of the Ca jump applied concurrently with T-tubule depolarization produced larger protein conformational change in the second phase with no appreciable effect on the first phase. Moreover, the Ca jump applied without depolarization produced only a Ca-dependent slow conformational change equivalent to the second phase of the depolarization-induced conformational change described above. All of these suggest the concept that the protein conformational change in the first phase represents the initial voltage-dependent activation of JFP, while that in the second phase the secondary activation by the released Ca.

The effective size of Ca jump for the production of protein conformational change in its second phase was in the submicromolar range, as determined in the experiment of Fig. 2. In contrast, the actual changes in the Ca concentration during the initial phase of calcium release, determined in the reaction solution (Fig. 3C), are in the range of a few nM, which seems to be too small to account for the large feedback effects on the calcium release kinetics observed here. This would indicate that the Ca that is actually affecting the release kinetics is not the Ca in the reaction solution, but a submicromolar Ca jump that is occurring in some compartmentalized region, the most likely location of such a compartment being in the cytoplasmic portion of the JFP. As a matter of fact, the recently published three-dimensional images of the JFP tetrameric complex show a discernible internal pocket in the T-tubule-side view (see Fig. 3b in (37) and Fig. 6a in (38) ). We postulate that the released calcium will generate a large Ca jump within this pocket because of its small internal volume, and that it effectively activates the JFP. This would be followed by dilution of the transiently compartmentalized Ca into the large volume of the reaction solution; the Ca value determined in the reaction solution was therefore very small. According to Stern(39) , (i) it would be extremely difficult to buffer [Ca] in the vicinity of a channel pore, even with a fast buffer like BAPTA, because the diffusion of the released calcium away from the pore is a very rapid non-equilibrium process, and (ii) even very high concentrations of BAPTA (e.g. 100 mM) would not perform a sufficient control of the [Ca] in the close vicinity of the pore. The first point is consistent with the discussion concerning the compartmentalized calcium we have just made. However, the present observation that submillimolar to millimolar concentrations of BAPTA appear to be effectively controlling the Ca feedback effect ( Fig. 1and Fig. 3) is incompatible with the second point. Clearly, more work is required along these questions regarding, in particular, the distance of the effective calcium activation site from the channel (29) and its relation to the diffusion dynamics of released Ca as well as BAPTA.

Turning to the kinetics of calcium release, a decrease in the BAPTA concentration enhanced both the initial activation and the subsequent attenuation of calcium release, as shown here. This suggests that the released Ca re-activated SR calcium release via the protein conformational change mechanism (namely the second phase described above), which in turn was followed by spontaneous attenuation of calcium release. Interestingly, the faster the initial activation was, the faster the subsequent attenuation became, suggesting that the initial activation and the subsequent attenuation are tightly coupled processes.

All of the above observations may be explained by a relatively simple model shown in Fig. S1.

The main framework of the reaction sequences shown in the top and bottom rows is based upon the concept derived from our recent studies. According to it, the signal-induced calcium release from SR is mediated by a conformational change in the JFP, which is a prerequisite mechanism for activation of the Ca channel, and this mechanism is commonly operating for various types of calcium release (e.g. release induced by T-tubule depolarization(33) , polylysine(40, 41) , and Ca(32) ). The model implies that upon applying T-tubule depolarization the JFP undergoes a rapid conformational transition from a resting state (R) to another state with higher MCA fluorescence (*R), which in turn is followed by a third state (*R) to open the Ca channel, as shown in the top portion of the scheme (cf. Reaction 2 in (33) ). The bottom portion of the scheme implies that the resultant Ca of various magnitudes (occurring presumably in the internal pocket of the JFP) stimulates the JFP, which following the first activation phase induces a second phase of calcium release by mediation of further protein conformational change, forming **R state with an even higher MCA fluorescence level.

The scheme also contains additional reaction steps involving isomerization of the channel protein from the open state (*R or **R) to an ``attenuated'' state (*R or **R). The states *R and **R are suggested from the present observation that the fluorescence intensity of the JFP-bound MCA remains high at least during an early phase of calcium release attenuation (compare Figs. 1A and 3B), although it eventually returns to the original low fluorescence state. ()Since the higher MCA fluorescence state (*, **) represents an active conformation of the JFP(32, 33, 41) , this suggests that the observed attenuation of SR calcium release is probably due to some mechanisms other than inactivation of the release channel per se. Depletion of the releasable calcium in the SR store during the initial phase of SR calcium release is widely regarded as one of the major factors responsible for release attenuation (e.g.(11) ). In this case, the faster calcium release would result in a sooner depletion of the releasable calcium, and in turn result in a faster attenuation. We also propose that the faster calcium release will cause a faster Ca re-uptake by the SR Ca ATPase(42) , which in turn will lead to an increase in the rate of attenuation of calcium release. Thus, at least these two factors would account for the present observation that the initial activation and the subsequent attenuation of calcium release occur in a coupled manner. Further studies are required to characterize the mechanisms responsible for the release attenuation.

To test the above reaction model, the time courses of protein conformational change (Fig. 4A), calcium release (Fig. 4B), and d(calcium release)/dt (Fig. 4C) curves were constructed by simulation of the model. These simulated curves represent reasonably well the overall features of the effects observed in the present experiments. Note that in this simulation all of the parameters were kept unchanged, except for the concentration of BAPTA buffer. The fact that we can still see the acceleration of both initial activation and subsequent attenuation (cf. Fig. 4C) even without changing any reaction constants suggests that the initial activation and the subsequent attenuation of calcium release are controlled solely by the feedback Ca. An interesting consequence of this mechanism is that the control of both initial activation and subsequent attenuation by Ca occurs in a synchronized fashion.

Figure 4: Simulation of the feedback reaction model (Fig. S1) produces curves that are qualitatively similar to those obtained in the present stopped-flow experiments: A, protein conformational change; B, calcium release curve; C, d(calcium release)/dt curve. The arbitrary rate constants used for simulation were: k = 30, k = 0.3; k = 5, k = 0.05; k = 3, k = 0.3; k = 200, k = 2.0; k = 5, k = 0.05; k = 3, k = 0.3. Note that a variation only in the BAPTA concentration (i < ii < iii < iv) resulted in major changes in the curve shape observed in the present experiments.

According to the above mechanism, suppression of the initial Ca-dependent acceleration by using high concentration of calcium-chelating agents should inevitably lead to the suppression of the rate of subsequent attenuation. Thus, the present study provides a reasonable explanation for both of the apparently contradictory observations in the muscle fiber literature concerning the effects of high concentrations of calcium-chelating agents on the voltage-dependent Ca transient as outlined in the Introduction. However, these studies in the literature have been done in frog muscle fibers, while the present study is in the vesicular system isolated from rabbit. Therefore, the present information may not be immediately applicable to the fiber system at least in some quantitative details.

FOOTNOTES

*

This work was supported by National Institutes of Health Grant AR 16922 and a grant from the Muscular Dystrophy Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed: Boston Biomedical Research Institute, 20 Staniford St., Boston, MA 02114. Tel.: 617-742-2010; Fax: 617-523-6649.

()

The abbreviations used are: SR, sarcoplasmic reticulum; DHP, dihydropyridine; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; E-C coupling, excitation-contraction coupling; JFP, junctional foot protein; MCA, methylcoumarin acetamide; MES, 2-(N-morpholino)ethanesulfonic acid; PI buffer, buffer solution containing proteolytic enzyme inhibitors; SAED, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate; T-tubule, transverse tubular system.

()

M. Yano, R. El-Hayek, and N. Ikemoto, unpublished data.

ACKNOWLEDGEMENTS

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