(Received for publication, December 5, 1994; and in revised form, December 21, 1994)
From the
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
): R
*R
*R
.
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). ()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
(
F). The major findings are as follows. The rate of
F 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
F 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
F and
SR Ca
release. However, blocking of Ca
release by 2 mM Mg
had virtually no
effect on
F. 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.
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 (
F).
As seen, chemical depolarization produced a rapid increase in the
fluoresecence intensity. The rate constant, as well as the amplitude of
F, 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
F 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
F, 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
F 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
F (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.
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
) 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. (
)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.