(Received for publication, July 27, 1994; and in revised form, November 18, 1994)
From the
Troponin (Tn), containing three subunits: Ca binding (TnC), inhibitory (TnI), and tropomyosin binding (TnT),
plays a crucial role in the Ca
regulation of
vertebrate striated muscle contraction. These three subunits function
by interacting with each other and with the other thin filament
proteins. Previous studies suggested that the primary role of TnT is to
anchor the TnI
TnC complex to the thin filament, primarily through
its interactions with TnI and tropomyosin. We propose here a new role
for TnT. Our results indicate that, when TnT is combined with the
TnI
TnC complex, there is an activation of actomyosin ATPase that
is Ca
-dependent. To determine whether the latter
results from a direct effect of TnC on TnT or indirectly from an effect
of TnC on TnI which is transmitted to TnT, we prepared a deletion
mutant (deletion of residues 1-57) of TnI, TnI
(Sheng et al. (1992) J. Biol. Chem. 267,
25407-25413), which interacts with TnC but not TnT. Both wild
type (TnI
TnC
TnT) and mutant
(TnI
TnC
TnT) Tn complexes demonstrated
equivalent activity in the Ca
regulation of
actomyosin-S1 ATPase activity. Similarly, both TnI and TnI
could equally reconstitute TnI-depleted skinned muscle fibers.
Therefore, since TnI
does not interact with TnT, these
results suggest that TnT reconstitutes native Ca
sensitivity via direct interaction with TnC. Thus Ca
binding to TnC would have a dual role: 1) release of the ATPase
inhibition by TnI and 2) activation of the ATPase through interaction
with TnT.
Much is known about the Ca regulation of
striated muscle contraction by the thin filament proteins troponin (Tn) (
)and tropomyosin (Tm). Tn consists of three subunits, the
Ca
binding (TnC), inhibitory (TnI), and Tm binding
(TnT). In the Tn complex, TnT has been shown to interact primarily with
TnI, and TnC has been shown to interact primarily with TnI.
Ca
binding to TnC initiates a chain of events which
leads to changes in the interactions between the Tn subunits, Tm, and
actin which results in the attachment of myosin cross-bridges and the
resulting contraction (for review see Zot and Potter(1987) and Grabarek et al.(1992)). Previous studies have shown that one of the
major steps in the regulatory process is a change in the interaction
between TnI and TnC when Ca
binds to the
Ca
-specific regulatory sites (Potter and Gergely,
1975) of TnC which results in the dissociation of TnI from actin
(Potter and Gergely, 1974; Hitchcock, 1975).
Although there has been
some evidence for Ca-dependent interaction between
TnC and TnT (Pearlstone and Smillie, 1978, 1982; Heeley et
al., 1987; Zot and Potter, 1987), little functional significance
has been ascribed to it until recently (Pan and Potter, 1992).
Previously it was thought that the role of TnT was primarily to anchor
the TnI
TnC complex to the thin filament through the interaction
of TnT with both Tm and TnI. However, Pan et al.(1992) showed
that two different COOH-terminal isoform fragments (
and
) of
TnT affected the affinity of the Ca
-specific sites of
TnC differently, implying a possible physiological significance to this
interaction.
To explore this further, we have studied the role of
TnT in the regulation of actomyosin-S1 ATPase activity. It is known
that although TnI can inhibit actomyosin ATPase activity, and TnC can
neutralize this inhibition, TnT is required to reconstitute native
Ca-dependent regulation (Zot and Potter, 1987). We
demonstrate here that there is a Ca
-dependent
activation of the actomyosin-S1 ATPase that only occurs in the presence
of TnT. We also present evidence that this TnT-dependent ATPase
activation, utilizing a deletion mutant of TnI (TnI
)
which interacts with TnC but not TnT, results from a TnC
Ca
-specific site-dependent interaction between TnC
and TnT. This result suggests that TnT probably plays a direct role in
the Ca
regulation of the actomyosin ATPase activity.
Furthermore, these results combined with previous studies, argue
strongly for a dual role for TnC in muscle regulation. Ca
binding to the Ca
-specific sites of TnC would
result in TnC interacting with both TnI (dissociating TnI from actin,
thereby relieving inhibition) and with TnT (resulting in ATPase
activation), with Ca
dissociation reversing both
processes.
Figure 1:
The effects of WTn, RTn, and Tn on actomyosin-S1 ATPase activity: Tn concentration dependence (A) and Ca
dependence (B). A, three Tn complexes were formed, as described in the legend
to Fig. 4, containing RTnT and RTnC and either RTnI(RTn) or
WTnI(WTn) or TnI
(Tn
). The concentration
dependence of these three complexes was tested on actomyosin-S1 ATPase
in the presence of Tm. Conditions: 23 °C, 20 mM KCl, 4
mM MgCl
, 2 mM ATP, 25 mM MOPS,
pH 7.0, 1 mM EGTA, 1 µM myosin-S1, 7 µM actin, and 1 µM Tm. The concentration of Tn added is
indicated on the abscissa. The dotted line indicates
the level of inhibition of the actomyosin-S1
Tm ATPase activity in
the presence of 3 µM RTnI. The neutralization of this
inhibition by 2 µM RTnC at pCa = 5.0 is
shown by the star in a circle symbol. The dashed line represents basal activity. The solid symbols indicate the
presence of Ca
(pCa = 5.0), and the open symbols indicate a low concentration of Ca
(pCa = 8.0); circles = RTn, triangles = WTn, and the squares =
Tn
. The data represent the average of three experiments. B, the Ca
dependence was carried out under
the same conditions as in A except that the Ca
concentration was varied as described previously. The three Tn
complexes, RTn (open triangles), WTn (open circles),
and Tn
(closed circles) were present at a
concentration of 1.5 µM. The data were fitted to the Hill
equation: relative ATPase activity (%) =
[Ca
]
/([Ca
]
+ pK
); where pK is the midpoint (pCa
) and n is the Hill coefficient. The pCa
was 6.56 for RTn, 6.55 for WTn, and 6.50 for
Tn
. The n were 1.10, 1.06, and 1.01,
respectively. The ATPase activity is expressed as a percentage of the
difference between the velocity at pCa 5.0 and pCa
8.0. Each point is an average of six
determinations.
Figure 4:
Size exclusion chromatography of the
ternary complexes of WTnI or TnI and TnC and TnT. WTnI (A) or TnI
(B) were mixed with RTnT and
RTnC (1:1:1, molar ratio) in 6 M urea, 1 M KCl, 25
mM MOPS, pH 7.0, 1 mM CaCl
, and 1 mM DTT on ice for 60 min. The complexes were then dialyzed
consecutively against KCl solutions of 1 M, 0.75 M,
0.5 M, 0.3 M, and 0.1 M, each containing 25
mM MOPS, pH 7.0, 1 mM CaCl
, and 1 mM DTT prior to loading on a Sephacryl S-200 column equilibrated with
the same solution. The insets are 4-20% gradient
SDS-PAGE gels of the fractions indicated. Closed circles,
OD
.
Figure 2:
TnT affinity chromatography. WTnI and
TnI were equilibrated with the starting solution
(starting solution = 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 0.5 mM CaCl
, and 1 mM DTT),
and after loading, the RTnT affinity column (see ``Materials and
Methods'') was washed with this solution (A, tubes
1-12). This was followed by a linear gradient (0.15-1 M NaCl in SS: B, tubes 13-63) and then with a
solution containing 1 M NaCl and 6 M urea in starting
solution (C, tubes 63-80). Circles,
OD
; closed squares, conductivity. The peaks
indicated to contain WTnI (tubes 69-75) and TnI
(tubes 5-10) were demonstrated by SDS-PAGE and Western
blotting.
Figure 3:
Size exclusion chromatography of WTnI or
TnI and TnT. WTnI (A) or TnI
(B) were mixed with RTnT (1:1 molar ratio) in a solution
containing 6 M urea, 1 M NaCl, 25 mM MOPS,
pH 7.0, 0.5 mM CaCl
, and 1 mM DTT on ice
for 60 min. These mixtures were then dialyzed consecutively against
NaCl solutions of 1 M, 0.75 M, O.5 M, 0.3 M, and 0.2 M, each containing 25 mM MOPS, pH
7.0, 0.5 mM CaCl
, and 1 mM DTT. These
mixtures were then loaded on a TSK column (Phenomenex G2000 WS)
equilibrated with 0.2 M NaCl, 25 mM MOPS, pH =
7.0, 0.5 mM CaCl
, and 1 mM DTT. The insets on the figures are 15% SDS-PAGE gels representing the
indicated fractions. Closed circles,
OD
.
Fig. 1B shows the
Ca dependence of actomyosin-S1 ATPase activity as a
function of pCa. The measurements were made essentially the
same as in Fig. 1A except the free Ca
concentration was varied at a fixed ratio of Tn/Tm. All three Tn
complexes showed essentially the same Ca
dependence.
These results suggest that, even in the absence of an interaction
between TnI and TnT, that the activation properties of TnT are not
lost, implying a Ca
-dependent interaction between TnC
and TnT in the Tn
complex, as well as in the RTn and WTn
complexes.
Figure 5:
Effects of WTnIRTnC and
TnI
RTnC complexes on vanadate-treated CSM. A and B, CSM were prepared as described under
``Materials and Methods.'' Two solutions were used:
contracting solution (pCa 4) containing 10
M [Ca
], 5 mM [Mg
], 4 mM EGTA, 5 mM ATP, 20 mM creatine phosphate, 20 units of CPK, and 10
mM imidazole, pH 7.0, ionic strength = 150 mM,
and relaxing solution (pCa 8) which had the same composition
except [Ca
] = 10
M. After an initial test contraction (A and B) the CSM was treated with 10 mM sodium
orthovanadate in pCa 8 (see ``Materials and
Methods'') for 10 min and then washed with the pCa 8
solution. After maximum force was obtained in pCa 8, the
preparations were incubated with the WTnI
RTnC (A) or
TnI
RTnC (B) complexes (10 µM)
in pCa 8 solution. After maximal inhibition was achieved, the
effect of the pCa 4 solution was tested. C, the
Ca
dependence (see ``Materials and
Methods'') of these reconstituted CSM were tested (WTnI
RTnC, solid circles; TnI
RTnC, solid
triangles) and compared with untreated fibers (solid
circles). These data (average of three experiments) were fitted to
the Hill equation (see Fig. 1) and the pCa
and n for these are 5.74 (n = 2.46), 5.7 (n = 2.24), and 5.74 (n = 2.38),
respectively.
Fig. 5C illustrates the Ca dependence of the CSM before vanadate treatment and the vanadate
treated CSM reconstituted with either WTnI
RTnC or
TnI
RTnC. The results for all three measurements
were not significantly different and indicate that the original
Ca
sensitivity could be restored with either binary
complex.
The results presented in this paper suggest that TnT plays an
important role in the regulation of contraction that has not been
considered previously. Basically, TnT appears to activate actomyosin-S1
ATPase activity when it is present in Tn complexes. TnI alone fully
inhibits the actomyosin-S1 ATPase activity in the presence of Tm as has
been shown previously (Weeks and Perry, 1978; Perry et al.,
1972; Greaser and Gergely, 1973; Greaser et al., 1972).
Addition of TnC to this, fully reverses the ATPase activity to the
basal ATPase level seen in the absence of TnI (Fig. 1), the so
called neutralization of TnI's inhibitory activity (Perry et
al., 1972). Interestingly, the ATPase activity could not be
further stimulated by subsequent additions of TnC. However, in the
presence of Ca, the ATPase activity with intact Tn
was much higher than that seen with the TnI
TnC complex in the
presence of Ca
. This activation of the ATPase could
only be observed in the intact Tn complex and suggested that this
activation is a property of TnT that requires Ca
binding to the regulatory Ca
binding sites on
TnC.
If the above is true, then there are at least two possibilities
to account for this observation. Ca binding to TnC
may cause a direct interaction to occur between TnC and TnT, thereby
altering the conformation of TnT. This signal would be transduced to Tm
and/or actin in some way leading to the activation of ATPase activity.
Alternatively, Ca
binding to TnC, which is known to
change its interaction with TnI, could transmit information indirectly
through a conformational change in TnI that would change the
conformation of TnT, leading to ATPase activation. Since TnI interacts
with TnT it is difficult to sort out these two possibilities. To
overcome this, we reasoned that if we could eliminate the interaction
between TnI and TnT without interfering with either TnI's
inhibition or TnC neutralization capabilities, that we would be able to
distinguish between these two possibilities directly.
As the results
indicated, TnI retained both of these properties.
Although the binding between TnI and TnT has been observed in previous
studies (Zot and Potter, 1987; Hitchcock, 1982; Sheng et al.,
1990; Horwitz et al., 1979; Chong and Hodges, 1982; Pearlstone
and Smillie, 1982), the specific nature of this interaction has not
been well understood. Previous cross-linking and lysine reactivity
studies have suggested that the NH
-terminal region
(residues 40-78) of TnI probably interacts with TnT (Hitchcock,
1982; Chong and Hodges, 1982). Consistent with this, we found that
TnI
did not bind to TnT (Potter et al., 1993).
Previous studies (Sheng et al., 1992a, 1992b) utilizing
TnI, have revealed that there are at least three types of
interaction which occur between TnC and TnI: 1) one which is dependent
upon Ca
binding to the Ca
-specific
sites of TnC; 2) one which is dependent upon Ca
or
Mg
binding to the Ca
-Mg
sites of TnC; and 3) one which is metal-independent. TnI
was shown to retain the Ca
-specific
site-dependent interaction but lost the
Ca
-Mg
site-dependent interaction
and exhibited a weaker metal independent interaction. Similar
conclusions have been made by Farah et al.(1994). In spite of
this difference from WTnI, TnI
retained full actomyosin
ATPase inhibitory activity that could be completely neutralized by TnC
(Perry et al., 1972; Weeks and Perry, 1978; Sheng et
al., 1992a, 1992b). Taken together, these results, and those
presented in this paper, suggest that the NH
terminus of
TnI may play a crucial role in stabilizing the interaction between not
only TnC and TnI, but also between TnT and TnI and is probably of
fundamental importance in maintaining the structural integrity of the
Tn complex.
We reasoned that if the ATPase activation seen with
intact Tn could still be observed in Tn complexes formed with
TnI, that this would imply a direct effect of TnC on TnT
since there would presumably be no interaction between TnI and TnT in
the ternary complex. Interestingly, the TnI
complex could
only be formed in the presence of Ca
, as might be
expected, since TnT could no longer bind to TnI and unless complexed
with TnC, TnT would precipitate under the conditions of this
experiment. Although the latter interpretation seems plausible, it is
not possible at present to completely exclude the possibility that the
interaction between TnC and TnI
may result in an
interaction between TnI
and TnT in the complex, that
would not occur between TnI
and TnT alone. We are
currently carrying out additional experiments to unequivocally decide
between these two possibilities.
Previous studies have shown that
TnT binds to TnC in a Ca-dependent manner (Zot and
Potter, 1987; Pearlstone and Smillie, 1978, 1982; Pan and Potter, 1992;
Heeley et al., 1987). The COOH-terminal half of TnT probably
contains the major binding site for TnC (Zot and Potter, 1987;
Pearlstone and Smillie, 1978; Pan and Potter, 1992), whereas the
NH
-terminal regulatory domain of TnC may contain the TnT
interaction site (Grabarek et al., 1981; Leavis and Gergely,
1984). Recent cross-linking studies have further defined the
interacting sites as including the residues 175-178 of TnT and a
region in the vicinity of Cys-98 of TnC (Leszyk et al., 1988).
Interestingly, this region of TnC also interacts with TnI in the
presence of Ca
(Leavis and Gergely, 1984; Leszyk et al., 1990). These observations suggest the possibility that
both TnI and TnT participate in the Ca
signal which
is transmitted by TnC. It is also possible that both TnI and TnT
interact in the same region or in close proximity on TnC.
The
ability of the Tn complex to regulate actomyosin ATPase
activity and contraction suggests that the sensitivity and
cooperativity of Ca
regulation were not affected
either by a lack of an interaction between TnI and TnT or by the loss
of the Ca
-Mg
-dependent interaction
between TnI
and TnC shown previously (Sheng et
al., 1992a, 1992b).
The actomyosin and CSM results strongly
suggest an important role for TnT in the regulation of contraction. Our
results indicate that the Ca-dependent activation of
actomyosin ATPase activity only occurs in the presence of TnT, even in
the absence of an interaction between TnI (TnI
) and TnT.
Increasing evidence indicates that TnT plays an important modulatory
role in the Ca
regulation of striated muscle
contraction. Several studies have established correlations between TnT
isoform composition and the Ca
sensitivity of force
production by skinned skeletal and cardiac muscle fibers (Schachat et al., 1987; Reiser et al., 1992). Tobacman and
Lee(1987) observed a difference in Ca
sensitivity
between reconstituted actomyosin systems regulated by two isoforms of
bovine cardiac TnT. We recently found that bacterially expressed rabbit
skeletal TnT fragments representing two carboxylterminal isoforms of
TnT exhibited different affinities for TnC and that there was a
significant difference in the Ca
affinity of the
regulatory sites of TnC in the binary complexes formed between the two
TnT isoform fragments and TnC (Pan and Potter, 1992). These results are
consistent with our hypothesis in this paper, that in addition to
interacting with TnI, TnC interacts directly with TnT in the
Ca
sensitive activation of contraction.
The way in
which TnC interacts with TnT and activates contraction is not well
understood at this time. As mentioned above,
Ca-dependent interactions between TnC and TnT are
thought to occur between the NH
terminus of TnC and the
carboxyl terminus of TnT. The carboxyl terminus of TnT (residues
159-259) has also been shown to interact with Tm in the vicinity
of Cys-190. In the presence of Ca
, the interaction
between the T2 fragment of TnT (residues 159-259) is strengthened
with TnC and weakened with Tm (Pearlstone and Smillie, 1983). In the
absence of Ca
the reverse is true. The interaction of
the NH
terminus of TnT with Tm in the vicinity of the Tm
overlap region has been shown to be insensitive to Ca
(Pearlstone and Smillie, 1982). Thus Ca
binding
to the Ca
-specific sites of TnC may produce an
interaction between the NH
terminus of TnC and the carboxyl
terminus of TnT that causes a dissociation or weakening of the
interaction between the carboxyl terminus of TnT and Tm, resulting in
the activation of the actomyosin ATPase, perhaps through a derepression
of the ATPase activity when TnT changes its interaction with Tm,
thereby altering the conformation of actin and its interaction with
myosin.
Our previous results (Pan and Potter, 1992) suggest that the
region coded by the variable carboxyl-terminal exon (amino acid
residues 221-233) in TnT may be important in the interactions
mentioned above, since the two carboxyl-terminal TnT fragments
containing either the or
exon exhibited different
affinities for Tm and also affected the Ca
affinity
of the Ca
-specific sites of TnC differently. Work is
in progress in our laboratory to further define these possibilities.
In summary, results presented here have suggested a new role to TnT
in the Ca regulation of contraction and a dual role
for TnC. In the absence of Ca
, TnI would be bound to
actin, inhibiting actomyosin ATPase activation and the carboxyl
terminus of TnT would be bound to Tm. Upon muscle activation and
Ca
binding to the Ca
-specific sites
of TnC, TnC would interact with TnI at the
Ca
-specific site-dependent interaction site on TnI,
thereby dissociating TnI from actin, relieving the actomyosin ATPase
inhibition. Simultaneously, TnC would interact with the carboxyl
terminus of TnT weakening the interaction of this region of TnT with Tm
and thereby derepress the ATPase activity resulting in the activation
ascribed here to TnT.