(Received for publication, October 27, 1995; and in revised form, January 11, 1996)
From the
In order to study the role of the Ca-specific
sites (I and II) and the high affinity
Ca
-Mg
sites (III and IV) of TnC in
the regulation of muscle contraction, we have constructed four mutants
and the wild type (WTnC) of chicken skeletal TnC, with inactivated
Ca
binding sites I and II (TnC1,2-), site III
(TnC3-), site IV (TnC4-), and sites III and IV
(TnC3,4C-). All Ca
binding site mutations were
generated by replacing the Asp at the X-coordinating position
of the Ca
binding loop with Ala. The binding of these
mutated proteins to TnC-depleted skinned skeletal muscle fibers was
investigated as well as the rate of their dissociation from these
fibers. The proteins were also tested for their ability to restore
steady state force to TnC-depleted fibers. We found that although the
NH
-terminal mutant of TnC (TnC1,2-) bound to the
TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)),
it was unable to reactivate Ca
-dependent force. This
supports earlier findings that the low affinity Ca
binding sites (I and II) in TnC are responsible for the
Ca
-dependent activation of skeletal muscle
contraction. All three COOH-terminal mutants of TnC bound to the
TnC-depleted fibers, had different rates of dissociation, and could
restore steady state force to the level of unextracted fibers. Although
both high affinity Ca
binding sites (III and IV) are
important for binding to the fibers, site III appears to be the primary
determinant for maintaining the structural stability of TnC in the thin
filament. Moreover, our results suggest an interaction between the
NH
- and COOH-terminal domains of TnC, since alteration of
sites I and II lowers the binding affinity of TnC to the fibers, and
mutations in sites III and IV affect the Ca
sensitivity of force development.
Vertebrate skeletal muscle contraction is activated by the
binding of Ca to the low affinity Ca
binding sites of troponin-C (TnC), called the
Ca
-specific sites and designated as sites I and
II(1) . These sites are located in the NH
-terminal
domain of TnC, which is separated from the COOH-terminal domain of TnC
by a 31-residue single helix (D/E)(2, 3) . The
COOH-terminal region of TnC contains two high affinity Ca
binding sites designated as sites III and IV and referred to as
the Ca
-Mg
sites(1) . Sites
I and II bind Ca
specifically with K
3
10
M
, whereas sites III and IV bind
Ca
with K
2
10
M
and Mg
with K
2
10
M
. Under physiological conditions, in
relaxed muscle, sites III and IV of TnC are primarily occupied by
Mg
and can become partially saturated with
Ca
during contraction depending on the time course of
the [Ca
] transient(4) . While the
process of Mg
exchange for Ca
is
much too slow (k
8 s
) to
trigger muscle contraction, the kinetics of Ca
binding to the NH
-terminal domain sites I and II of
TnC are coupled with the rate of muscle activation, implying that these
sites are directly involved in muscle regulation. Inactivation of
either of them significantly decreases regulatory function of
TnC(5) , indicating that sites I and II are both required for
the full regulatory activity of TnC. In contrast, the role of the high
affinity Ca
-Mg
binding sites, III
and IV, is still not entirely clear. A number of studies have suggested
a structural role for these sites in maintaining the stability of the
whole troponin complex in the thin filament, presumably through TnC-TnI
interactions (1, 6) . The interaction interface of the
COOH-terminal domain of TnC containing the high affinity
Ca
-Mg
sites, III and IV, has been
shown to be located in the NH
-terminal region of TnI
(residues 1-98) and also near the NH
-terminal end of
the inhibitory region of TnI containing residues
96-116(7, 8, 9, 10) . This
so-called antiparallel fashion of TnC-TnI interaction is also true for
the Ca
-dependent interaction of the
NH
-terminal regulatory domain of TnC with the COOH-terminal
region of TnI as well as with the COOH-terminal end of the TnI
inhibitory peptide(10, 11, 12) .
Recently
we have shown that thrombin fragments of the NH-terminal
domain of TnC, containing Ca
binding sites I and II,
maintain the regulatory properties of intact TnC, whereas fragments
from the COOH-terminal domain are mostly involved in the
Ca
-Mg
-dependent interactions of TnC
with TnI(13) . In the present study we have examined the role
of the NH
- and COOH-terminal Ca
binding
sites of TnC using mutants of TnC that have either inactivated
Ca
-specific sites I and II (TnC1,2-) or
structural site III (TnC3-), IV (TnC4-), or III and IV
(TnC3,4-). The effect of these mutations on their structure and
function was investigated using the TnC-depleted skinned skeletal
muscle fiber system(5, 6) , where incorporation of the
TnC mutants and steady state isometric force measurements are readily
performed. We found that the NH
-terminal mutant of TnC
(TnC1,2-) was able to bind to the fibers but failed to develop
steady state force. Although TnC1,2- bound to the fibers in the
pCa 8 relaxing solution, it was easy to displace it with WTnC,
indicating a low affinity of this mutant for its binding sites in the
fibers. All three COOH-terminal mutants of TnC (TnC3-,
TnC4-, and TnC3,4-) demonstrated weakened ability to bind
to TnC-depleted fibers. They dissociated from the fibers with different
rates, implying that Ca
binding sites III and IV of
TnC contribute differently to the maintenance of the structural
integrity of the whole troponin complex. Consistent with our thrombin
fragment studies(13) , site III appears to be the primary
determinant of the affinity of this interaction between TnC and its
binding site in the fiber in the presence of Mg
. Once
bound to the fibers, all three of these mutants were able to activate
steady state force as in unextracted intact fibers.
The amino acid
sequence of the WTnC and all mutants used in this study contained
glutamic acid and aspartic acid at positions 99 and 100, respectively,
as originally incorporated in the synthesized cDNA of TnC (14) used in this study. Subsequently it was found that the
correct sequence of chicken skeletal TnC (16, 17) at
these positions consists of alanine (Ala) and asparagine
(Asn
), respectively. To test the significance of this
two-amino acid difference, the sequence of WTnC was corrected to
Ala
and Asn
, and the protein was tested in
the fiber experiments as described below. No difference was found
between the corrected and noncorrected WTnCs with respect to steady
state force development of WTnC reconstituted fibers (data not shown).
The mutated proteins were expressed in Escherichia coli BL-21 and purified based on Sheng (5) with the following
modifications. One ml of a log phase subculture of E. coli was
inoculated into 2 liters of enriched medium. After 12 h of culture,
protein expression was induced with 0.4 mM isopropyl
1-thiol--D-galactopyranoside, and the cells were
harvested 4 h later. Cells pelleted by centrifugation (5 min at 6400
g) were resuspended in 1M NaCl, 50 mM Tris, pH 7.5, 0.1 mM CaCl
, 1 mM dithiothreitol with 1 µM leupeptin and pepstatin and
10 µM phenylmethylsulfonyl fluoride. Alternatively, cells
were stored at this point at -20 °C for later isolation of
protein or sonicated (for 6 min at high intensity in 30-s pulses with
90-s rests) on wet ice with a sonicator (Heat Systems model XL2020).
Sonicates were cleared by centrifugation, adjusted to 1 M ammonium sulfate, and if a precipitate formed, centrifuged again.
Cleared supernatants were applied to a 2.5
20-cm
phenyl-Sepharose column, equilibrated in the above buffer containing 1 M ammonium sulfate (to increase the affinity of the expressed
protein to the hydrophobic matrix), washed with the same buffer, and
then eluted with 50 mM Tris, pH 7.5, 2 mM EDTA, 1
mM dithiothreitol. Protein fractions were dialyzed against a
solution containing 50 mM Tris, 1 mM CaCl
, 6 M urea, pH 8.0, and applied to a 5
8-cm DE-52 column. The column was washed with the same buffer,
and protein was eluted with a salt gradient of 0-500 mM KCl (2
300 ml). Fractions of >95% purity (judged by
SDS-PAGE) (
)were pooled and dialyzed against 5 mM ammonium bicarbonate, lyophilized, and stored at -70 °C.
Both the unextracted and the reconstituted fibers were
also tested for force development in solutions of increasing
[Ca], from pCa 8 to pCa 4. Data were
analyzed using the following equations: 1) percentage of force restored
= 100
(force restored after reconstitution with TnC
- residual force), and 2) percentage of change in force =
100
[Ca
]
/([Ca
]
+
[Ca
]
), where
``[Ca
]'' is the free
Ca
concentration that produces 50% force and n is the Hill coefficient.
Figure 1:
Binding of Ca to WTnC, TnC1,2-, TnC3-, TnC4-, and
TnC3,4-. The binding study was performed using the equilibrium
dialysis method (see ``Materials and Methods''). WTnC bound
3.8 ± 0.17 mol of Ca
/mol of protein, whereas
TnC1,2-, TnC3-, TnC4-, and TnC3,4- bound 2.27
± 0.11, 2.93 ± 0.02, 2.87 ± 0.33, and 2.14
± 0.27, respectively. Data are the average of three
experiments.
Figure 2:
SDS-PAGE of WTnC, TnC1,2-,
TnC3-, TnC4-, and TnC3,4-. Samples of the purified
mutants of TnC were run on 15% SDS-PAGE in the presence of 1 mM CaCl (+) (lanes 2, 4, 6, 8, and 10) or 2 mM EGTA(-) (lanes 1, 3, 5, 7, and 9).
WTnC (lanes 1 and 2), TnC1,2- (lanes 3 and 4), TnC3- (lanes 5 and 6),
TnC4- (lanes 7 and 8), and TnC3,4- (lanes 9 and 10).
Figure 3:
Simultaneous force (A) and
fluorescence (B) measurements of skinned skeletal muscle
fibers reconstituted with AC-TnC1,2-. After initial steady state
force measurement of the skinned fibers (A), endogenous TnC
was extracted with 5 mM EDTA, pH 7.8 (see ``Materials and
Methods''). The extracted fibers were then incubated with a
fluorescently labeled mutant of TnC having inactivated Ca binding sites I and II (AC-TnC1, 2-). Reconstitution was
performed in pCa 8, and incorporation of the protein was detected
utilizing fluorescence measurements (B). Steady state force
was not changed as compared with the residual force of the extracted
fibers (A). Inactivation of the regulatory Ca
binding sites (I and II) of TnC resulted in a loss of force
restoration (A). There was a small change in fluorescence
intensity upon Ca
binding to AC-TnC1,2- (B). The fibers were then exposed to WTnC, and the force and
fluorescence measurements were repeated. Restoration of steady state
force was about 90% compared with the initial force. The fluorescence
intensity decreased to 10% as a result of the replacement of AC-TnC1,2-
by WTnC.
Figure 4:
The
concentration dependence of force restoration in TnC-depleted skinned
fibers reconstituted with WTnC, TnC3-, TnC4-, and
TnC3,4-. Skinned skeletal fibers were tested for initial steady
state isometric force and then extracted with 5 mM EDTA, pH
7.8, for 30 min. After TnC extraction, the fibers were incubated with
increasing concentrations (abscissa) of WTnC (),
TnC3- (
), TnC4- (
) or TnC3,4- (
)
dissolved in the pCa 8 solution for 15 min. Individual fibers were used
for each concentration tested. Excess unbound protein was removed by
washing in the pCa 8 solution followed by the measurement of force
development in the pCa 4 solution. For TnC3,4- two curves are
presented. The extra curve (
) represents measurements with the
protein present in the pCa 4 contraction solution. Data points are the
average of three to six experiments.
Fig. 5illustrates the binding properties of WTnC,
TnC3-, TnC4-, and TnC3,4- to TnC-depleted fibers. The
experimental protocol is presented for TnC3- in Fig. 5A. The fibers, initially tested for force
development in the pCa 4 solution, were extracted with 5 mM EDTA for 30 min. The residual force approximated the amount of
endogenous TnC remaining in the fibers. TnC-depleted fibers were then
reconstituted with TnC3- with a 20-min incubation, followed by
force measurements to confirm that the maximal level of reconstitution
had been achieved. Then steady state isometric force was measured at
different time intervals to determine the rate of TnC3-
dissociation from the fibers. Between the measurements the fibers were
relaxed in the pCa 8 solution. The same protocol was used for WTnC and
all TnC mutants. Fig. 5B demonstrates the time course
of the dissociation of all of the mutated proteins and WTnC from the
reconstituted fibers. As illustrated, WTnC and TnC4- exhibited
similar slow dissociation from the fibers, while the dissociation of
TnC3- was much faster. After 30 min of exposure of the
TnC3- reconstituted fibers to the pCa 8 solution, 50% of the
restored force was lost. The double mutant, TnC3,4- was the
fastest dissociating protein, showing very low affinity for the fibers
when incubated in the absence of Ca
(presence of
Mg
). Isometric force dropped to the residual level
after a 25-min incubation of the TnC3,4- reconstituted fibers in
the pCa 8 solution (Fig. 5, B and C). After a
1- h exposure of the reconstituted fibers to the relaxing solution,
they were tested to determine their ability to regain force when
reconstituted with WTnC. These fibers were able to bind WTnC, and force
was restored to
80-90% of the value of unextracted fibers (Fig. 5C). Thus the observed drop in force was due to
TnC dissociation and not due to the deterioration of the fibers with
time.
Figure 5:
Dissociation rates of WTnC, TnC3-,
TnC4-, and TnC3,4- from TnC-depleted skinned muscle fibers
reconstituted with the mutated TnCs. A, the protocol for the
dissociation rate measurements of the TnC3- reconstituted fibers. A-C, skeletal muscle fibers were tested for force
development in the pCa 4 solution, and then the endogenous TnC was
extracted with 5 mM EDTA, pH 7.8, for 30 min. Incorporation of
WTnC, TnC3-, TnC4-, and TnC3,4- into the fibers was
achieved with a 20-min incubation. B, steady state isometric
force was measured at different time intervals to determine the rate of
dissociation of the incorporated mutants from the fibers. , WTnC;
, TnC4-;
, TnC3-;
, TnC3,4-. C, after 1 h of dissociation of the reconstituted mutants from
the fibers, the fibers were incubated with WTnC dissolved in pCa 8
solution. Force restoration was then tested in the pCa 4 solution.
, WTnC;
, TnC4-;
, TnC3-;
,
TnC3,4-.
Figure 6:
The force-pCa relationship for fibers
reconstituted with WTnC, TnC3-, TnC4-, and TnC3,4-. Solid curves, the unextracted fibers () were tested for
force development in solutions of increasing Ca
concentrations. Dotted curves, after extraction of
endogenous TnC, the fibers (
) were reconstituted with WTnC (A), TnC3- (B), TnC4- (C), and
TnC3,4- (D) and tested for force development in the
different pCa solutions, from pCa 8 to pCa 4. The solid and dashed lines represent the best fit of the data to the Hill
equation (see ``Materials and Methods''). A,
control, pCa
= 5.46, n = 2.99;
after reconstitution with WTnC, pCa
= 5.48, n = 2.60; B, control, pCa
=
5.41, n = 3.32; after reconstitution with TnC3-,
pCa
= 5.57, n = 3.67; C,
control, pCa
= 5.46, n = 2.90;
after reconstitution with TnC4-, pCa
= 5.66, n = 3.15; D, control, pCa
= 5.47, n = 4.28; after reconstitution
with TnC3,4-, pCa
= 5.72, n =
4.19.
We studied the role of Ca specific sites (I
and II) and the high affinity Ca
binding sites (III
and IV) of TnC in the regulation of skeletal muscle contraction. Under
physiological conditions the structural sites (III and IV) are
saturated with Mg
, whereas sites I and II are not
occupied by metal. The initiation of muscle contraction occurs when
Ca
binds to the low affinity sites (I and II) in TnC
since exchange of Mg
for Ca
at
sites III and IV is too slow to account for muscle
activation(4) . Therefore, sites I and II of TnC are the
regulatory sites, whereas the high affinity Ca
binding sites (III and IV) are thought to maintain the structural
integrity of the whole troponin complex in the thin
filament(6, 25) .
Our results demonstrate that
NH-terminal sites I and II of TnC indeed play an important
role in the Ca
-triggered regulation of muscle
contraction. Skeletal muscle fibers reconstituted with the mutated TnC,
having inactivated sites I and II (TnC1,2-), were not able to
develop steady state force, although the mutant protein bound to the
TnC depleted fibers even in the absence of Ca
(presence of Mg
). This is in accord with our
previous study (5) where two separate mutants containing either
inactive site I or II could only partially restore force to
TnC-depleted skeletal fibers. Consistently, inactivation of the only
regulatory site in cardiac TnC resulted in the loss of regulation of
the Ca
-dependent ATPase activity in TnC-extracted
myofibrils(26) .
Interestingly, inactivation of the
regulatory sites in TnC also affected the binding of TnC1,2- to
the TnC-depleted fibers. Its binding affinity was lower than WTnC,
since WTnC was able to displace TnC1,2- from the fibers. This
suggests an interaction between two domains in TnC where altered
NH-terminal sites I and II have affected the structure and
function of the COOH-terminal sites III and IV. Since the binding of
TnC to the fibers is affected by the binding of Mg
or
Ca
to the Ca
-Mg
sites (see below and (6) ), it is possible that
inactivation of sites I and II lowers the metal binding affinity of
sites III and IV and consequently the affinity of TnC1,2- for the
fibers.
On the other hand, inactivation of either of the
COOH-terminal high affinity Ca binding sites (III or
IV) affected the association of the mutated TnCs from the thin
filament. All three mutated proteins, TnC3-, TnC4-, and
TnC3,4-, dissociated from the reconstituted fibers faster than
WTnC, with the TnC3,4- dissociating the fastest. Interestingly,
sites III and IV appeared not to be equal in terms of maintaining the
structural stability of TnC in the fibers. The mutant of TnC that
contained inactivated Ca
binding site IV
(TnC4-) bound to the TnC-depleted fibers and restored force
almost as well as the wild type TnC. Its dissociation rate from the
reconstituted fibers was also similar to the rate of dissociation of
WTnC. The active Ca
binding site III in this protein
was therefore sufficient to maintain its Mg
-dependent
binding to the TnC-depleted fibers. In contrast to that, the mutant
containing inactivated Ca
binding site III
(TnC3-) dissociated from the reconstituted fibers much faster
than TnC4-. After 30 min exposure of the TnC3-
reconstituted fibers to the pCa 8 solution, only half of the
reincorporated protein remained bound to the fibers. The observed
difference in the ability of these two sites (III and IV) to maintain
the structural integrity of TnC in the fibers does not appear to be due
to the different Ca
binding stoichiometry of the
mutated proteins. Both, TnC4- and TnC3- bound essentially 3
mol of Ca
/mol of protein, respectively, as compared
with the 4 mol of Ca
bound by WTnC. They also bound
to the TnC-depleted skinned fibers in a Mg
-dependent
manner over the same concentration range as WTnC. The observed
difference in the rate of their dissociation from the fibers is in
accord with our previous investigation on the thrombin fragments of
TnC, demonstrating the functional differences between the
Ca
binding sites III and IV(13) , with the
site III being more crucial for maintaining the structural integrity of
the troponin complex.
The distinction between sites III and IV of
TnC was also observed by Brito (22) in cardiac TnC (CTnC)
mutants having either inactive Ca binding site III or
IV. CTnC mutants containing inactivated site III produced greater
instability in the C-terminal domain of CTnC than mutants containing
inactivated site IV, as was judged by NMR spectroscopy. Our mutant with
inactivated Ca
binding site III (TnC3-) was
also less stable in terms of its association with the thin filament in
the fiber and dissociated from the reconstituted fibers faster than the
mutant with inactivated site IV (TnC4-). Moreover, the
Ca
-induced difference of the electrophoretic
migration of the mutated proteins in the presence of SDS was lost
following inactivation of Ca
binding site III
(TnC3-) or III and IV (TnC3,4-). In contrast, the mutant
TnC4- with site III still active demonstrated a small
Ca
-sensitive change in the electrophoretic migration.
The greatest Ca
-dependent mobility difference was
observed for WTnC and TnC1,2-. Apparently, inactivation of the
NH
-terminal sites I and II in TnC does not affect the
structural change needed to produce this effect of Ca
binding to sites III and IV. These results suggest that in the
presence of Ca
the NH
-terminal region of
TnC is more stable than its COOH terminus, and inactivation of two high
affinity Ca
binding sites (III and IV) significantly
decreased the structural stability of TnC, preventing the change in
migration of TnC3,4- on SDS-PAGE due to Ca
.
In another study, no functional difference between sites III and IV
in CTnC was found with respect to the affinity of the mutated proteins
for the thin filament(23) . This might be due to functional
differences that have been found between skeletal and cardiac muscle
preparations (23, 26) , differentiating the affinity
of the TnC isoforms to the thin filament and possibly altering their
interaction with the native TnI. However, consistent with our
observation, the double mutant with both Ca binding
sites III and IV inactivated was unable to restore force unless it was
present in the contraction solution(23) . No difference between
the dissociation rate of chicken skeletal TnC mutants containing either
inactivated site III or IV and reconstituted to rabbit TnC-depleted
fibers was observed by Sorenson et al.(24) . This may
be due to the very fast dissociation rate of both mutated TnCs found by
these authors in contrast to the rates detected in our study for TnC3-
or TnC4-. After a 10-min exposure of the reconstituted fibers to the
Mg
-containing relaxing solution, they observed as
much as 70% of the tension decrease for both mutated TnCs(24) .
In contrast, comparable rates of dissociation were only observed by us
for the double TnC mutant containing inactivated sites III and IV
(TnC3,4-). This mutant demonstrated a significant inability to
remain bound to the fibers in the presence of Mg
and
could restore steady state force only when it was present in the
contraction (pCa 4) solution. The reason for this difference between
our work and that of Sorenson et al.(24) remains
unknown but could be due to their use of trifluoroperazine in their TnC
extraction solution, which may interfere with subsequent TnC rebinding
or to the fact that their preparation likely has a lower amount of
endogenous TnC than ours. Interestingly, we observed that the force-pCa
relationship was shifted toward lower Ca
concentrations for all of our mutated TnCs, whereas in Sorenson et al.(24) only their equivalent TnC3- mutant
showed an altered tension-pCa relationship, with a shift toward higher
Ca
concentrations. The increased Ca
sensitivity of force development produced by our mutants suggests
that the COOH-terminal high affinity Ca
binding sites
III and IV may affect the Ca
binding and regulatory
properties of the NH
-terminal low affinity Ca
binding sites I and II. This was not observed by Negele et
al.(23) where their wild types and mutants of cardiac
TnCs, when reconstituted into TnC-depleted skeletal myofibrils showed
no change in the Ca
sensitivity of ATPase activity.
Our data clearly demonstrate that all four Ca binding sites of TnC are important for the physiological function
of TnC in the thin filaments. Sites I and II are responsible for the
Ca
-dependent activation of muscle contraction, and
inactivation of Ca
binding to these sites completely
abolishes the activation of muscle. Sites III and IV are important for
the structural stability of TnC in the whole troponin complex, with
site III being more critical for TnC association with the fibers. Site
IV appears not to be essential for the structural stability of TnC
within the thin filament, similar to our observations in our thrombin
fragment studies(13) . The occupancy of site III by
Mg
appears to be sufficient to maintain the
association of TnC4- with the other troponin subunits and prevent
its dissociation from the fibers. Thus, Ca
binding
sites III and IV do not appear to contribute equally to metal-dependent
TnC binding to the thin filament via its COOH-terminal domain.
Additional studies will be necessary to distinguish between these sites
in terms of the Ca
/Mg
-dependent
interactions of TnC with TnI and other proteins in the thin filament.
Moreover, the present results suggest an interaction between the two
domains of TnC where the structural alterations within the
NH
-terminal domain affect the function of its COOH-terminal
region and vice versa. Inactivation of sites III and IV
affected the Ca
sensitivity of force development,
whereas mutations of sites I and II lowered the binding affinity of TnC
to the fibers.