©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conformational Changes in the Junctional Foot Protein/Ca Release Channel Mediate Depolarization-induced Ca Release from Sarcoplasmic Reticulum (*)

(Received for publication, December 5, 1994; and in revised form, December 21, 1994)

Masafumi Yano (1) Roque El-Hayek (1) Noriaki Ikemoto (1) (2)(§)

From the  (1)Boston Biomedical Research Institute, Boston, Massachusetts 02114 and the (2)Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In an attempt to monitor the kinetic events occurring in the junctional foot protein (JFP) during excitation-contraction coupling, the JFP moiety of isolated triads was covalently labeled in a site-directed manner with methylcoumarin acetate (MCA) using a recently developed technique (Kang, J. J., Tarcsafalvi, A., Carlos, A. D., Fujimoto, E., Shahrokh, Z., Thevenin, B. J. M., Shohet, S. B., and Ikemoto, N.(1992) Biochemistry 31, 3288-3293). Chemical depolarization of the transverse tubular system (T-tubule) moiety of labeled triads after appropriate priming induced first a rapid increase of the fluorescence intensity of the JFP-bound MCA probe, and then sarcoplasmic reticulum (SR) Ca release. Upon increasing the magnitude of T-tubule depolarization by increasing the degree of ionic replacement, both the amplitude of the MCA fluorescence change and the amount of released Ca increased in parallel. Blockers of T-tubule-to-SR communication, such as nimodipine and low concentration of neomycin, inhibited both the MCA fluorescence change and the SR Ca release. In contrast, the release blocking concentration of Mg (2 mM) inhibited only SR Ca release without affecting the fluorescence change. These results suggest that upon T-tubule depolarization the original state of the JFP (R) isomerizes to an activated state with higher MCA fluorescence (*R), which in turn changes into a subsequent state in which the release channel is open (*R(o)): R *R *R(o).


INTRODUCTION

The electromechanical coupling model proposed by Chandler et al. (1) about 2 decades ago can be interpreted in terms of recently resolved molecular components in the following way (for reviews, see (2, 3, 4, 5, 6, 7, 8) ). The excitation signal elicited in the T-tubule is sensed by the dihydropyridine receptor (DHPR). (^1)The signal is then transmitted to the neighboring JFP/Ca release channel protein, producing conformational changes in the JFP. This in turn activates the Ca channel in the JFP(9, 10, 11) , and SR Ca release ensues. The interaction between the two major proteins may be mediated by a direct contact via the cytoplasmic loop of the DHPR (12, 13, 14) or by a third protein playing an intermediary role(15) .

According to our recent study(16) , the fluorescence intensity of the conformational probe MCA, which was incorporated into the JFP moiety in a site-specific manner, showed almost perfect parallelism to the activation/inhibition profile of SR Ca release by Ca and ryanodine. This suggested that the JFP conformational change may play an important role in the regulation of various types of Ca release.The main goal of this study is to investigate the hitherto merely hypothetical JFP conformational change during excitation-contraction (E-C) coupling by means of the MCA probe technique (16) and of newly devised methods to produce voltage-controlled SR Ca release in vitro(17) . Here we report that chemical depolarization of the T-tubule moiety of the triad produces a rapid increase in the fluorescence intensity of the JFP-attached MCA probe (DeltaF). The major findings are as follows. The rate of DeltaF is much faster than that of Ca release from the SR, indicating that the fluorescence change precedes Ca release. Upon increasing the degree of ionic replacement, and thus the magnitude of T-tubule depolarization, both DeltaF and the subsequent SR Ca release increased in a parallel fashion. The DHP receptor antagonist nimodipine (cf. (18) ) or a novel blocker of the T-tubule-to-SR communication neomycin (19) inhibited both DeltaF and SR Ca release. However, blocking of Ca release by 2 mM Mg had virtually no effect on DeltaF. These results suggest that depolarization-induced SR Ca release is mediated by a conformational change of the JFP, which is a prerequisite of the activation of the Ca release channel.


EXPERIMENTAL PROCEDURES

Preparation

The triad-enriched microsomal fraction (triad) was prepared from rabbit leg and back muscle by differential centrifugation as described previously(17, 20) . After the final centrifugation, the sedimented fraction was homogenized in a solution containing 0.3 M sucrose, 0.15 M potassium gluconate, proteolytic enzyme inhibitors (0.1 mM phenylmethanesulfonyl fluoride, 10 µg/ml aprotinin, 0.8 µg/ml antipain, 2 µg/ml trypsin inhibitor) and 20 mM MES, pH 6.8 (PI buffer), to a final protein concentration of 20-30 mg/ml. The preparation was quickly frozen in liquid nitrogen and stored at -70 °C.

Site-directed Labeling of the Junctional Foot Protein with the Fluorescent Conformational Probe

Specific fluorescent labeling of the JFP moiety of the triad was performed using the cleavable hetero-bifunctional cross-linking reagent, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED; (16) ) with the aid of neomycin as a site-specific carrier as follows. First, neomycin-SAED conjugates were formed by incubating 0.4 mM neomycin with 0.2 mM SAED in 20 mM HEPES (pH 7.5) for 15 min at 22° in the dark. The reaction was quenched by a 10-fold dilution with 10 mM lysine. Fifty µl of neomycin-SAED conjugate (final neomycin concentration was 2 µM) were mixed in the dark with 1 mg of triad protein, brought to 1 ml with PI buffer, and centrifuged for 15 min at 100,000 times g. The sedimented fraction was resuspended in 1 ml of PI buffer and photolyzed with UV light in a Pyrex tube at 4 °C for 10 min. beta-Mercaptoethanol was added (100 mM final) to cleave the disulfide bond of SAED. After incubation on ice for 1 h, the mixture was again centrifuged for 15 min at 100,000 times g, and the sedimented triads were resuspended in 50 µl of PI buffer to a final protein concentration of 20 mg/ml. The MCA incorporation was also done using polylysine as a site-directing carrier as described previously(16) . In agreement with our previous report, these procedures (both neomycin- and polylysine-mediated incorporation) resulted in the specific incorporation of the MCA into the JFP moiety of the triad as determined by fluorometry of electrophoretically separated protein bands (data not shown).

Induction and Monitoring of Depolarization-induced Ca Release

To depolarize the T-tubule moiety of the triad, we used the Na-replacement protocol that had been originally used to produce T-tubule-mediated tension development in the skinned fiber system (21, 22) and more recently adapted to the isolated triad system(17) . The fluorescently labeled triads (1.6 mg/ml) were incubated in the priming solution (Solution A) containing 150 mM potassium gluconate, 15 mM NaCl, 5 mM MgbulletATP, an ATP-regenerating system (2.5 mM phosphoenolpyruvate, 10 units/ml pyruvate kinase), 100-150 µM CaCl(2), 20 mM imidazole (pH 6.8) at 22 °C for 6 min or longer. Then, 15 µl of Solution A was mixed with 135 µl of Solution B containing various concentrations of potassium gluconate, sodium gluconate, 15 mM NaCl, 2.5 µM fluo-3, 20 mM imidazole (pH 6.8). Major ionic compositions of the priming Solution A and the depolarizing Solution B for each degree of ionic replacement (G1, G5.5, and G10; defined in the legend to Table 1) are given in Table 1. Inhibitors, when used, were included in Solution B. The time courses of SR Ca release induced at various levels of depolarization were monitored with a stopped-flow fluorometer (BioLogic SFM-3 with MOS-200 optical system) using fluo-3 as a Ca probe (excitation at 437 nm, emission at 530 nm with a 510-nm cut-off filter). Approximately 30-40 traces of the fluo-3 signal were averaged for each experiment.



Fluorescence Assays of the Protein Conformational Change

To monitor the time course of depolarization-induced changes in the fluorescence intensity of the JFP-bound MCA probe, the fluorescently labeled triads (2.4 mg/ml) were incubated in Solution A, and 15 µl of the primed triads was mixed with 135 µl of Solution B devoid of fluo-3. 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 various degrees of depolarization were monitored with the same stopped-flow fluorometer. As a control, the fluorescently labeled triads were incubated in Solution A devoid of ATP (neither polarization of the T-tubule nor active Ca loading of the SR), and the same mixing as above was performed. The control trace was subtracted from the traces of the polarized and depolarized sample. Approximately 70-90 traces of the MCA signal were averaged for each experiment.


RESULTS

We produced various degrees of depolarization of the T-tubule moiety of the MCA-labeled triad using the previously described Na-replacement protocol (see (17) and ``Experimental Procedures''). The time courses of (a) depolarization-induced changes in the fluorescence intensity of the JFP-bound MCA probe and (b) depolarization-induced Ca release from SR were monitored using stopped-flow fluorometry (Fig. 1). Although the Ca release rate was slightly reduced after labeling, the voltage-dependent changes in the Ca release kinetics (Fig. 1, rightpanel) show a pattern identical to that of the unlabeled triads described in our recent report(17) . Thus, there was no Ca release on dilution without ionic replacement (Fig. 1, G1). As the degree of ionic replacement and the concomitant T-tubule depolarization were increased, both the rate and the magnitude of Ca release also increased. Fig. 1also shows the corresponding time courses of the depolarization-induced changes in the fluorescence intensity of the JFP-attached MCA probe (DeltaF). As seen, chemical depolarization produced a rapid increase in the fluoresecence intensity. The rate constant, as well as the amplitude of DeltaF, increased again in a proportional fashion to the degree of ionic replacement, suggesting that rapid conformational change of the JFP reflected by the MCA fluorescence change is a phenomenon that is tightly coupled with both T-tubule depolarization and Ca release. The rate constant of DeltaF is significantly higher than that of Ca release at each magnitude of T-tubule depolarization (cf. Table 2and Table 3). Based on these results we propose that the depolarization signal transmitted from the T-tubule would produce first conformational changes in the JFP as manifested in DeltaF, and subsequently activation of the release channel as illustrated in the following hypothesis.


Figure 1: Time courses of the fluorescence increase of the JFP-bound MCA probe (leftpanel) and SR Ca release (rightpanel) induced by various degrees of T-tubule depolarization. After priming the MCA-labeled triads in Solution A, the T-tubule moiety was chemically depolarized to various extents by dilution with Solution B of different compositions (cf. Table 1). Changes in the fluorescence intensity of the protein-bound MCA and Ca release from the SR moiety were monitored in the stopped-flow fluorometric system as described under ``Experimental Procedures.'' Note that the time scales of the left and rightpanels differ by a factor of 2. To facilitate the comparison of the kinetics of the MCA fluorescence change and Ca release, the MCA fluorescence curves shown in the leftpanel are retraced in the rightpanel. G1, no ionic replacement (no T-tubule depolarization); G5.5, an intermediate ionic replacement for intermediate degree of depolarization; G10, a maximal ionic replacement for maximal depolarization under our current stopped-flow conditions.







This hypothesis was further investigated through the following experiments. Nimodipine is one of the specific blockers of the DHP receptor/voltage sensor protein(23) . Neomycin is another potent blocker of the T-tubule-to-SR communication, as evidenced by our recent finding that a low concentration of neomycin (0.1 µM) completely blocked Ca release induced by chemical depolarization without affecting Ca release induced by direct stimulation of the JFP moiety by polylysine(19) . As shown in Fig. 2, either nimodipine (10 µM) or neomycin (0.1 µM) blocked both DeltaF and SR Ca release after depolarization, presumably by blocking Step 1 above. These results indicate that both the JFP conformational change and the subsequent SR Ca release are under the control of the signal transmitted from the T-tubule. The results also suggest that blockage of the conformational change inevitably inhibits the subsequent release-activating step. Interestingly, 2 mM Mg inhibited Ca release with virtually no effect on DeltaF (Fig. 2). This suggests that inhibition by several mM Mg of the Ca channel opening (24, 25) occurs without preventing conformational change (namely in Step 2), confirming the concept that the JFP conformational change precedes the activation of SR Ca release channel.


Figure 2: Blockers of the dihydropyridine receptor (10 µM nimodipine) and T-tubule-to-SR communication (0.1 µM neomycine) inhibit both MCA fluorescence change and SR Ca release, while SR channel blocker (2 mM Mg) inhibits only Ca release. The primed MCA-labeled triads were chemically depolarized to maximum extent (G10) with Solution B containing various inhibitors as indicated (the concentration indicated in the text represents the final concentration of the inhibitor after mixing Solution B with Solution A). Then, the time courses of the MCA fluorescence change and SR Ca release were recorded as described in the legend to Fig. 1. The reference traces represent the time courses of MCA fluorescence change (leftpanel) and Ca release (right panel) obtained with no added inhibitors, respectively.




DISCUSSION

The two major events in the skeletal muscle E-C coupling are (i) sensing of the membrane potential change (depolarization) by the DHPR of the T-tubule and (ii) activation of the SR Ca release channel that resides in the JFP. However, little is known about how these events are coupled. In attempt to resolve the intermediate reaction steps involved in this coupling process, we incorporated the fluorescent conformational probe MCA into the JFP moiety of isolated triads in a site-directed manner and then investigated in parallel the conformational response of the JFP to the depolarization signal and the induced Ca release from SR. The main findings are as follows. (a) The protein conformational change precedes SR Ca release. (b) The voltage-dependent activation pattern of Ca release is an identical copy of that of protein conformational change, suggesting that the mode of regulation of SR channel is already determined by the preceding protein conformational change. (c) Blockage of the signal transmission from the T-tubule to SR by nimodipine or neomycin inhibited the protein conformational change and, in turn, Ca release; this suggests that both protein conformational change and Ca release are induced by the signal derived from the T-tubule. (d) Inhibition by Mg of the opening of the Ca release channel (24, 25) had virtually no effect on the protein conformational change, suggesting that the Mg block of channel opening occurred after protein conformational change. From these results, we conclude that upon stimulation of the JFP via T-tubule, the JFP undergoes a transition from a state of lower MCA fluorescence (R) to another state (*R) of higher MCA fluorescence, which in turn is followed by a third state (*R(o)) to open the channel.

Low concentration of neomycin (e.g. 0.1 µM) blocks only depolarization-induced Ca release(19) , but at higher concentrations (several micromolar) it blocks other types of Ca release as well, such as release induced by polylysine(16) , caffeine(26) , and Ca(26, 27) . Thus, 0.1 µM neomycin works as a specific blocker of the signal transmission from T-tubule to SR(19) , but at several micromolar it works as a channel blocker similar to Mg and ruthenium red. Interestingly, no fluorescent labeling occurred when 0.1 µM neomycin was used as a carrier of SAED, and to obtain an appreciable level of specific labeling of the JFP the concentration of the carrier had to be increased to several µM, e.g. 2 µM, as done in the present study. Thus, the voltage-dependent fluorescence signal described in this study must have derived from the MCA probe attached to the channel-blocking neomycin-binding domain.

As reported previously(16) , the fluorescence intensity of the MCA probe incorporated into either the neomycin-binding or polylysine-binding domain of the JFP increased with increasing concentrations of Ca in parallel to the activation of SR Ca release. Furthermore, the JFP-specific Ca release trigger polylysine induces an increase in the fluorescence of the MCA probe attached to either the neomycin or the polylysine domain, again paralleling the dose-dependent activation of SR Ca release by polylysine. (^2)Together with the present finding that the kinetics of the JFP conformational change parallel the T-tubule depolarization, we propose that the conformational-switch mechanism proposed in (R *R) operates, as a common mechanism of channel activation, regardless of the type of Ca release trigger (Ca, polylysine, or T-tubule depolarization).

All the data shown in this paper were obtained with samples MCA-labeled at the neomycin-binding domain. According to our preliminary data, samples labeled at the polylysine-binding domain also show essentially the same voltage-dependent changes in MCA fluorescence as described here. Since neomycin and polylysine appear to bind to different domains (16) , the JFP conformational change induced by these release triggers would be rather global.

In conclusion, the present results suggest that the coupling between the T-tubule signal and SR Ca release is mediated by a rapid conformational change of the JFP. The present study has opened a new way to resolve the intermediary reaction steps involved in the signal transmission/transduction processes. In order to further characterize the protein conformational change described here, however, several important problems remain to be investigated. These include the localization of the site of probe attachment in the primary and quaternary structures of the JFP and characterization of the structural significance of the observed fluorescence increase.


FOOTNOTES

*
This work was supported by Grant AR 16922 from the National Institutes of Health 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.

(^1)
The abbreviations used are: DHPR, dihydropyridine receptor; DHP, dihydropyridine; JFP, junctional foot protein; MCA, methylcoumarin acetamide; MES, 2-(N-morpholino)ethanesulfonic acid; SR, sarcoplasmic reticulum; SAED, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate; T-tubule, transverse tubular system; E-C, excitation-contraction.

(^2)
R. El-Hayek, M. Yano, and N. Ikemoto, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Michel Ronjat and Yves Dupont (Laboratoire de Biophysique Moléculaire et Céllulaire, Centre d'Etudes Nucleaires, Grenoble, France) for testing the MCA sample with the Biologic illumination system before purchase and Dr. John Gergely for comments on the manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.