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First published online November 30, 2007
Journal of Experimental Biology 210, 4399-4410 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.008722
Unphosphorylated twitchin forms a complex with actin and myosin that may contribute to tension maintenance in catch
1 Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507,
Japan
2 Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293,
Japan
3 Muscle Biology Group, University of Arizona, Tucson, AZ 85721,
USA
4 Graduate School of Agricultural and Life Sciences, The University of
Tokyo, Bunkyo, Tokyo 113-8567, Japan
Author for correspondence (e-mail:
awatabe{at}mail.ecc.u-tokyo.ac.jp)
Accepted 18 September 2007
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Summary |
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Key words: catch contraction, twitchin, ABRM, myosin, actin
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Introduction |
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;
Twarog, 1976). Secretion of
serotonin abolishes the catch state by increasing intracellular [cAMP] and
activation of cAMP-dependent protein kinase (PKA). These various phases
(Watabe and Hartshorne, 1990)
can be reproduced in skinned catch muscle fibers, i.e. an increase in
[Ca2+] initiates contraction, a decrease in [Ca2+]
promotes catch and addition of cAMP terminates the catch state.
Since the discovery of catch there has been considerable interest in
defining the molecular basis responsible for force maintenance with very low
energy expenditure. Early ideas suggested that two types of linkages were
formed, the catch linkage consisting of paramyosin–paramyosin
interactions and the contractile linkages of actin and myosin (Ruegg, 2001;
Watabe and Hartshorne, 1990).
In this scheme, phosphorylation of paramyosin by PKA would reduce or eliminate
the paramyosin association and in the presence of Ca2+ allow
cross-bridge cycling. Subsequently, the paramyosin hypothesis became less
popular and catch was suggested to reflect attached non-cycling
cross-bridge–actin interactions, the so-called `linkage' hypothesis
(Lowy et al., 1964). It was
suggested that phosphorylation of the myosin rod was the critical regulatory
step (Castellani and Cohen,
1987). It was found subsequently that the only protein
phosphorylated by PKA in ABRM (catch) fibers was the high molecular mass
protein, twitchin (Siegman et al.,
1997). These and subsequent studies
(Siegman et al., 1998;
Funabara et al., 2001a)
proposed that twitchin is an integral component of the catch mechanism and is
a phosphorylation-dependent regulator of the catch state. Dephosphorylated
twitchin promotes catch and phosphorylation of twitchin by PKA releases catch.
From in vitro studies, involving isolated thick and thin filaments
under relaxing conditions, it was further suggested that the only components
necessary for catch were actin, myosin and twitchin
(Yamada et al., 2001). As a
working hypothesis it was assumed that catch reflected attached non-cycling
cross-bridges that bear the mechanical load and whose formation is regulated
by phosphorylation of twitchin (Butler et
al., 2001). But, other observations made it necessary to
reconsider a basic tenet of the `linkage' hypothesis. Galler and coworkers
found that catch depends on cAMP- and pH-sensitive linkages but not on the
cross-bridge–actin interactions
(Galler et al., 2005). It has
also been suggested that the mechanical link in catch could be the interaction
between dephosphorylated twitchin and actin, where phosphorylation of twitchin
abolishes interaction (Shelud'ko et al.,
2004). Binding of synthetic myosin filaments to F-actin filaments
and dephosphorylated twitchin has been shown, but not direct binding of
twitchin to actin (Yamada et al.,
2001). The idea that alternative links may be involved was further
developed and although regulation of catch by twitchin phosphorylation is
accepted there is considerable evidence to support the idea that the
mechanical links in catch are not attached cross-bridges
(Andruchova et al., 2005;
Hopflinger et al., 2006;
Butler et al., 2006).
The Mytilus ABRM twitchin is composed of a single polypeptide of
530 kDa containing multiple repeats of immunoglobulin (Ig) and fibronectin
type III motifs and a single kinase domain
(Funabara et al., 2003) and is
very similar to twitchin and twitchin-related proteins from other species
(Funabara et al., 2005).
In vitro phosphorylation assays with PKA indicate that ABRM twitchin
incorporates 3 mol phosphates mol–1 twitchin
(Funabara et al., 2001a). Two
of the three phosphorylation sites are referred to as D1 and D2 and are
located at S1075 and S4316 near the N and C termini, respectively. It was
suggested that both sites are involved in the regulation of catch contraction
in vivo (Funabara et al.,
2003).
In light of the suggestion that the mechanical links in catch involve interactions other than the force-generating actin–myosin complex, this study was initiated to examine interactions with twitchin, with an emphasis on effects mediated by phosphorylation at the D2 site. Because of the high mass of twitchin (and complexity in binding assays) a D2 peptide (composed of the D2 site plus two Ig motifs) was used. Important observations were that the dephosphorylated D2 peptide binds to myosin and F-actin and promoted complex formation between myosin and F-actin. In addition the localization of twitchin on myofilaments of ABRM was examined. These cumulative data are interpreted to indicate that the D2 site of twitchin mediates interaction between myosin and F-actin.
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). ABRM actomyosin was prepared as previously reported from
Mytilus galloprovincialis Lamarck
(Funabara et al., 2001b).
Chicken skeletal actomyosin was prepared by the same method as that for ABRM
actomyosin. Myosin was prepared from chicken fast skeletal and scallop
Patinopecten yessoensis Jay striated muscles
(Stafford et al., 1979).
Paramyosin was isolated from ABRM (Watabe
et al., 1989). Actin was prepared from acetone powder of chicken
fast skeletal muscle (Mommaerts,
1951). Twitchin was prepared as described
(Funabara et al., 2001a).
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Mg2+-ATPase assay
ATPase assays were carried out in 20 mmol l–1
3-(N-morpholino)propanesulfonic acid (MOPS)-NaOH (pH 7.4) containing
30 mmol l–1 KCl, 2 mmol l–1
MgCl2, 1 mmol l–1 ATP, 80 µg
ml–1 actomyosin, 16 µg ml–1 D2 peptide
mutant and 0.1 mmol l–1 CaCl2 or 1 mmol
l–1 ethyleneglycol bis(2-aminoethylether)tetraacetic acid
(EGTA). ATPase of chicken fast skeletal actomyosin was also assayed in the
presence of 80 µg ml–1 ABRM paramyosin. Liberated
inorganic phosphate was determined by the Malachite Green method
(Kodama et al., 1986).
Solid-phase binding assay
Solid-phase binding assay was carried out according to published methods
(Weitkamp et al., 1998) with
some modifications. In this assay, washing, blocking reaction, dilution of
samples and detection were carried out using reagents included in Protein
Detector ELISA kit (KPL, Gaithersburg, MD, USA). Wells of an enzyme-linked
immunosorbent assay (ELISA) microplate were coated with 100 µl of 10 µg
ml–1 scallop striated adductor myosin, ABRM paramyosin or
chicken fast skeletal F-actin in 1x Coating Solution included in the kit
for 1 h at room temperature. After blocking, 100 µl of 10 µg
ml–1 TWD2-S or thiophosphorylated TWD2-S were hybridized in
1x Coating Solution for 1 h at room temperature with proteins coated on
the plate. Anti-6xHis and anti-mouse IgG antibodies conjugated with alkaline
phosphatase were used, respectively, as the primary and secondary antibodies.
The reaction was traced by measuring absorbance at 630 nm after the substrate
solution included in the kit was added to wells.
Cosedimentation binding assay
30 µg ml–1 scallop striated adductor myosin, 300 µg
ml–1 chicken fast skeletal F-actin and 10 µg
ml–1 TWD2-S were mixed in 20 mmol l–1
MOPS-NaOH (pH 7.4), containing 30 mmol l–1 KCl, 4 mmol
l–1 MgCl2, 1 mmol l–1 ATP and 1
mmol l–1 EGTA, on ice for 30 min and centrifuged at 3000
g. The supernatants and precipitates obtained were subjected
to SDS-PAGE.
Identification of twitchin binding region of actin
Chicken fast skeletal actin was extensively digested by trypsin in 20 mmol
l–1 Tris-maleate (pH 7.0) containing 50 mmol
l–1 KCl and 10 mmol l–1 CaCl2 for
24 h at 37°C. The digests were subjected to reverse-phase high-performance
liquid chromatography with a TSKgel ODS-80T column (4.6 mmx15 cm)
(Tosoh, Tokyo, Japan) and adsorbed peptides were eluted with 0%–40%
acetonitrile linear gradient. The isolated peptides were subjected to the
solid-phase binding assay using TWD2-S as a probe. The peptide bound to TWD2-S
was sequenced using the Applied Biosystems Procise 492 HT protein sequencer
(Applied Biosystems, Foster City, CA, USA).
Competitive binding assay
A peptide AGFAGDDAP synthesized based on the sequence A21–P29 of
chicken fast skeletal actin was subjected to competition binding assay for
TWD2-S according to the method of the solid-phase binding assay using the same
ELISA kit. Wells of a microplate were coated with 100 µl of 50 µg
ml–1 chicken fast skeletal F-actin in 1x Coating
Solution. After washing and blocking, 100 µl of 5 µg
ml–1 TWD2-S was hybridized with the adsorbed F-actin for 1 h
at room temperature. After washing, 100 µl of 0, 50, 100 and 150 µg
ml–1 synthetic peptide were added to the wells and hybridized
in 1x BSA Diluent/Blocking Solution included in the kit for 1 h at room
temperature. Detection was performed as described in the solid-phase binding
assay. Assays were done in the absence of added Ca2+.
Production of anti-TWD2-S antibody and its specificity
Anti-twitchin D2 antibody used for electron microscopy was raised against
TWD2-S in rabbit. The specificity of the antibody to twitchin was confirmed by
immunoblotting. To confirm whether anti-TWD2-S antibody used in the present
study reacts only to twitchin in ultrathin sections and isolated thick
filaments, immunoblotting analysis was carried out. Myofibrillar proteins were
electroblotted onto a polyvinylidene difluoride (PVDF) membrane after
separation on SDS-PAGE. The membrane was hybridized with anti-TWD2-S antibody
for 1 h at room temperature and reacted with anti-rabbit IgG antibody
conjugated with horseradish peroxidase as the secondary antibody after
washing. Detection was carried out using 0.2 mg ml–1
3,3'-diaminobenzidine tetrahydrochloride and 0.005%
H2O2.
To examine the reactivity of the antibody to twitchin and its
phosphorylated form, immunoblotting analysis and ELISA using TWD2-S and its
phosphorylated form were performed. TWD2-S was phosphorylated as described
previously (Funabara et al.,
2001a). Immunoblotting was performed as described above except for
twitchin peptides. ELISA was carried out using the same kit used in the
solid-phase binding assay in the present study. Wells of a microplate were
coated with 10, 20 and 30 µg ml–1 twitchin peptide, TWD2-S
or its phosphorylated form, and anti-TWD2-S antibody was added to the wells
after blocking. Anti-rabbit IgG antibody conjugated with alkaline phosphatase
was used as the secondary antibody. All solutions for ELISA were included in
the kit.
Electron microscopy
Ultrathin sections for electron microscopy were prepared from ABRM fibers
fixed with 4% paraformaldehyde (pH 7.2) in active contraction raised by 1 mmol
l–1 acetylcholine, catch state provoked by subsequent washing
with artificial seawater, and relaxation after treatment with 1 µmol
l–1 serotonin. These contraction–relaxation stages were
monitored by tension measurement. Fibers showed maximum tension at 50 s after
treatment with 1 mmol l–1 acetylcholine (at 25°C), and at
this point were fixed with paraformaldehyde and represented fibers in the
active state of contraction. Paraformaldehyde-fixed fibers were dehydrated
with ethanol and treated with Lowicryl K4M resin. Ultrathin sections of about
80 nm were placed on Ni-150 meshes covered with collodion membranes, treated
with the anti-twitchin D2 primary antibody at 0.1 mg ml–1 for
1 h, and then with AuroProbe EM goat anti-rabbit IgG conjugated with colloid
gold as the secondary antibody for 30 min each on ice. The ultrathin sections
thus prepared were stained with 1% uranyl acetate and 1% lead citrate each for
10 min at room temperature, and observed using a JEOL JEM 2000EX transmission
electron microscope.
ABRM thick filaments for electron microscopy were prepared according to
Nonomura's method (Nonomura,
1974). Briefly, ABRM relaxed by 1 µmol l–1
serotonin was treated with 0.05% saponin and the resulting skinned fibers were
homogenized on ice in an appropriate volume of 10 mmol l–1
phosphate buffer (pH 6.8) containing 0.1 mol l–1 KCl, 5 mmol
l–1 MgCl2 and 10 mmol l–1 ATP.
The homogenate containing thick and thin filaments was mixed with the
anti-twitchin D2 antibody at 0.1 mg ml–1 for 5 min on ice,
followed by reaction with the above secondary antibody conjugated with colloid
gold for 5 min on ice. The filaments were negatively stained with 1% uranium
acetate on Ni-400 meshes. A part of the negatively stained preparation was
rotary shadowed with platinum, using a BAF060 freeze-etch machine (BAL-TEC AG,
Liechtenstein).
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Binding to myosin, actin and paramyosin of twitchin D2 peptides
It has been claimed that intact twitchin binds to both myosin
(Yamada et al., 2001) and
actin (Shelud'ko et al.,
2004). Thus, the binding of actin, myosin and paramyosin to the
TWD2 constructs was investigated using a solid-phase binding assay (see
Materials and methods). Thiophosphorylated TWD2-S was used as a probe instead
of TWD2-D, because the thiophosphorylated TWD2-S mimicked the phosphorylation
form more effectively than TWD2-D. For convenience (i.e. higher yield) we
prepared chicken fast skeletal actin and scallop striated adductor myosin.
Fig. 3A,C,E show typical
results in the solid-phase binding assay for unphosphorylated TWD2-S and
thiphosphorylated TWD2-S with actin, myosin and paramyosin, respectively.
Unphosphorylated TWD2-S bound to each of the proteins, but not to the
thiophosphorylated construct. Relative binding abilities of 10 µg
ml–1 twitchin D2 peptides are shown in
Fig. 3B,D,F. These results
clearly indicate that the twitchin D2 site interacts with actin, myosin and
paramyosin in a phosphorylation dependent-manner.
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)
(Fig. 4D). To investigate
whether this region of actin (D26–D27) might be involved in binding to
the D2 peptide a synthetic peptide, A21–P29, was prepared and shown to
bind to the unphosphorylated TWD2-S but not the thiophosphorylated form (data
not shown). This synthetic peptide competed with actin for binding to
unphosphorylated TWD2-S (Fig.
4E). The cumulative evidence indicates that L10–P29, exposed
on the surface of actin, is involved in interaction with twitchin
(Fig. 4F).
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Formation of a complex of myosin, actin and TWD2-S
An essential feature of the catch mechanism is the maintenance of tension
at low intracellular [Ca2+] where myosin is not active. Do myosin
and actin maintain a connection with twitchin during catch? To address this
question, myosin and actin were mixed with unphosphorylated TWD2-S under low
[Ca2+] and high [ATP] to mimic catch conditions. Due to the
requirements for relatively large amounts of myosin and F-actin, scallop
striated adductor and chicken fast skeletal muscle were used as starting
materials, respectively. The binding of myosin, actin and unphosphorylated
TWD2-S was assayed under conditions mimicking catch using low-speed
sedimentation at low ionic strength where myosin is insoluble and actin
soluble (Fig. 5A). Initially,
an excess of unphosphorylated TWD2-S or thiophosphorylated TWD2-S was used
(molar ratio for twitchin construct:myosin of 50:9). Thiophosphorylated TWD2-S
had no effect on the distribution of actin in the supernatant fraction
(Fig. 5A). Cosedimentation of
each of the three components was detected only when unphosphorylated TWD2-S
was used. Subsequently, it was found that lower ratios of unphosphorylated
TWD2-S:myosin also promoted cosedimentation. Even at a ratio of 1:150
(unphosphorylated TWD2-S:myosin) a trimeric complex formation was detected
(Fig. 5B). It is proposed that
the in vivo molar ratio of twitchin:myosin in catch muscle is about
1:15 (Siegman et al., 1997).
These results suggest that under catch conditions the unphosphorylated D2
peptide mediates interaction between myosin and F-actin.
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Transmission electron microscopy was employed to localize twitchin in ultrathin sections of ABRM using the anti-D2 peptide antibody. Different experimental conditions with ABRM were adopted to simulate active contraction, catch state and relaxed state. The abundance of gold particles was highest for ABRM in the catch state (Fig. 7C,D) followed by a decreased distribution in the contracted state (Fig. 7A,B) and a further reduction for the relaxed state (Fig. 7E,F). These data, summarized in Table 2, suggest that twitchin in the catch state is unphosphorylated. Gold particles, which were observed only around thick filaments and not thin filaments, appeared marginally separated from the thick filament surface in active and catch states (Fig. 7B,D), while gold particles in the relaxed state were closer to the thick filaments (Fig. 7F). This slight shift of gold particles indicates that the location of the twitchin D2 site may move at different stages of muscle contraction. No gold particles were observed when ABRM was treated in the absence of the antibody (Fig. 7G,H).
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) and
separated into a mixture of multiple normal distributions
(Fig. 8A). The distribution
showed a periodicity of 36.25 nm and the estimated slope of the regression
line (36.29) through the origin (Fig.
8B) was not significantly different from the observed period
(F-test, P=0.91). This interval of 36.25 nm on the thick
filaments is half of the 72.5 nm periodicity of paramyosin filaments
(Squire, 1981).
Furthermore, electron microscopy of isolated ABRM thick filaments reacted
with the anti-D2 peptide antibody demonstrated that gold particles conjugated
with the secondary antibody were located periodically on the surface of the
thick filaments (Fig. 9A),
confirming the above observations with ultrathin sections. In fact the
homogenate of ABRM prepared in the relaxed state contained both thick and thin
filaments, but only thick filaments were selected and subjected to analysis.
Gold particles attached to the secondary antibody also were associated with
granular structures having an average diameter of 20.2 nm, i.e. about the
diameter of the myosin head (Fig.
9B), suggesting an interaction between the D2 peptide and the
myosin head. Also noted were ultrathin structures of over 50 nm in length and
3.8 nm diameter that extended along the axis of the thick filaments
(Fig. 9C). These dimensions
suggest, tentatively, that the 50 nm structures may be part of twitchin
filaments, extending along the surface of the thick filament. Resolution was
not adequate to obtain more accurate estimates of the filament length. ABRM
twitchin as observed by rotary shadowing is an elongated molecule
approximately 225 nm in length with a spherical head
(Fig. 9D), as reported
elsewhere (Vibert et al.,
1993). The antibody against the kinase domain
(Funabara et al., 2001a)
reacted to this head region (Fig.
9D). [For these experiments the antibody to the kinase domain was
used since its titer for twitchin was higher than the D2 peptide antibody
(data not shown).] Models of the parallel array of twitchin molecules (red)
are superimposed on the Bear-Selby net pattern
(Bear and Selby, 1956) and
shown relative to the myosin head distribution (blue)
(Fig. 9E). The D2 site is
located adjacent to the kinase domain at its C-terminal edge
(Fig. 1A) and thus may be
positioned close to the myosin head.
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). Thus it
is expected that binding of either myosin or twitchin to this N-terminal
region of actin would be competitive. However, this competition would occur
only with unphosphorylated D2 peptide. The binding site for the D2 peptide on
myosin remains to be determined, but the electron microscopy data suggests an
interaction with the myosin head. As emphasized, the D2 peptide interaction
with myosin is phosphorylation-dependent, and it is not known if other
phosphorylation-independent links are formed between intact twitchin and the
contractile apparatus. The finding that twitchin remains attached to thick
filaments isolated under various conditions suggest that there are other
twitchin–thick filament interactions (with myosin and/or paramyosin). We
propose that a critical feature of the catch mechanism is the
phosphorylation-dependent interaction of the twitchin D2 region with both
F-actin and myosin. This complex does not involve cross-bridge–actin
interactions. The locations of the actin-binding and the myosin-binding sites
on the D2 peptide are not known. It is assumed that distinct sites are
involved (possibly in the two flanking Ig motifs) and that both are regulated
by phosphorylation of D2. It is not necessary that direct binding to the D2
site (S4316) is involved, but this could occur for one of the sites. In
addition to twitchin there are several muscle proteins that contain Ig motifs
[e.g. titin, myosin light chain kinase (MLCK), telokin and the myosin-binding
proteins (MyBPs)] and the ability of certain Ig motifs to bind to myosin has
been documented. For example, in cardiac MyBP-C the interaction with myosin
was mapped to the C-terminal Ig motif
(Alyonycheva et al., 1997).
Subsequently, it was found that the unphosphorylated MyBP-C motif bound to the
S2 region of myosin and influenced cardiac mechanics. Interestingly,
phosphorylation of the MyBP-C motif by PKA abolished these effects by
eliminating binding to myosin (Kunst et
al., 2000). In cardiac muscle the stoichiometry of MyBP-C to
myosin heads is about 1:8 (Kunst et al.,
2000). The binding of Ig motifs to actin is not well documented,
although the N-terminal two Ig motifs of long MLCK have been implicated in
actin binding (Yang et al.,
2006).
These results are consistent with those of other investigators (see
Introduction) and, for example, may explain the myofilament interconnections
proposed by Andruchova et al. (Andruchova
et al., 2005). The competition between the unphosphorylated D2
peptide and loop 2 of myosin for the binding site on actin also may be
pertinent to the results of Butler et al. regarding the low- and high-force
states (Butler et al., 2006). A
higher affinity of myosin for actin in the high-force state would effectively
displace the twitchin–actin interaction. This is consistent with the
observation (Butler et al.,
2006) that activation of the catch muscle by Ca2+
increases the proportion of myosin in the high-force state and decreases
interaction of twitchin with actin. Transition to the low-force state, by
decreasing Ca2+ levels, would favor the twitchin–actin
interaction. It should be emphasized that a direct role for Ca2+ in
binding of the D2 peptide to either actin or myosin is not suggested and there
is no evidence from our data to implicate a Ca2+ sensitive step,
other than the regulation of actin-activated ATPase by Ca2+ binding
to myosin. Dephosphorylation of twitchin by calcineurin
(Castellani and Cohen, 1992;
Yamada et al., 2004) would be
coincident with initiation of contraction since both events are
Ca2+ dependent, and thus for most of the contractile phase twitchin
is dephosphorylated and theoretically available to compete with myosin for
actin. In practice, twitchin does not inhibit the in vivo contraction
of ABRM fibers in the presence of Ca2+
(Siegman et al., 1998). A
competition between twitchin and myosin might explain the in vitro
inhibition of Mg2+ ATPase activity of actomyosin by relatively high
concentrations of TWD2-S.
Results from the localization of twitchin on the thick filament indicate
that the twitchin molecules are distributed on the surface of the filament at
a periodicity of 36.25 nm, half of the 72.5 nm periodicity of paramyosin
filaments (Squire, 1981). It
is suggested that the D2 site at the C-terminal end of twitchin aligns with
myosin heads, i.e. at half of the 72.5 nm axial periodicity
(Cohen, 1982). In the model
proposed in Fig. 9E the
twitchin molecule could extend through about three of the 72.5 nm repeats or
nodes, if arranged parallel to the filament axis. The myosin rod extends
axially through two nodes. Thus there is a possibility of other interactions
between twitchin and the myosin rod. The molar ratio of twitchin to myosin is
about 1:15 in ABRM catch muscle (Siegman
et al., 1997). Therefore, even if all of the twitchin molecules
interact with myosin, the number of trimeric complexes is small compared with
total myosin. It is not known if there are cooperative effects on neighboring
myosin molecules induced by the binding of twitchin to myosin. The ultrathin
filaments observed above on the surface of the thick filaments are thought to
be twitchin and these reflect the underlying Bear-Selby net
(Bear and Selby, 1956) (see
Fig. 9C).
There are several aspects of the catch mechanism to be resolved. In an
earlier report (Funabara et al.,
2003) it was stated that phosphorylation of both the D1 and D2
sites is required for relaxation from the catch state. The D1 site is unusual
in that relatively high levels of phosphorylation (40–50%) are found
while catch is maintained. The D2 site is more sensitive to effects of
phosphorylation; low levels of phosphorylation are found during catch and high
levels of phosphorylation accompany the release from catch
(Funabara et al., 2003). The
more marked phosphorylation dependence of the D2 site prompted this present
study, but the role of the D1 site is not understood and should be resolved by
further studies. Another area for future study is to identify the
phosphorylation-dependent binding site on myosin for the D2 peptide and also
to determine if other interactions between native twitchin and myosin occur.
If present, these could anchor the twitchin molecule to the surface of the
thick filament. Based on the in vitro results of Yamada et al.
(Yamada et al., 2001) it is
assumed that paramyosin is not an essential component of the catch mechanism,
but whether it has any influence on catch under in situ conditions
remains to be determined. The phosphorylation-dependent binding of the D2
peptide to paramyosin is shown above. It is known that in fibers of molluscan
smooth muscle the catch state is sensitive to pH
(Hopflinger et al., 2006) and
is reduced on moderate alkalinization to pH 7.2–7.7, i.e. at those pH
values used for the binding experiments described above. It is important to
determine if any of the interactions between isolated proteins show a marked
pH dependence and this can be tested experimentally. On the other hand, the
sensitivity to pH may be associated only with the intact contractile system.
Recently, it was found that striated muscles from oyster and scallop contain
twitchin and this regulates interaction between thin and thick filaments at
low [Ca2+] (Tsutsui et al.,
2007), as it does in molluscan catch muscle. These results suggest
a similar role for twitchin in striated and smooth molluscan muscle and the
molecular scheme outlined above may be applicable to both muscle types.
In summary, our results provide novel data on the molecular interactions involved in catch. In Fig. 10A the different phases of the contractile cycle in catch muscle are shown, with associated changes in [Ca2+], tension and twitchin phosphorylation. Starting with relaxed muscle (Fig. 10B), twitchin is phosphorylated (by PKA) and the D2 peptide does not interact with either actin or myosin. It is assumed that in the relaxed state there are no interfilament connections. Stimulation by acetylcholine increases [Ca2+], which promotes contraction (by binding to and activating myosin) and activates calcineurin. Twitchin is dephosphorylated but does not bind to actin (although theoretically this interaction is still possible) because of competition with the increased population of high-force states, i.e. the cross-bridge–actin interaction predominates. The onset of catch follows a reduction in [Ca2+] to close to resting levels and inactivation of myosin. The low-force state (the detached cross-bridge being part of the low-force state) allows the D2 twitchin region to bind to F-actin and myosin and form a mechanical force-bearing complex. In this model there is no direct effect of Ca2+ on the binding of the unphosphorylated D2 peptide to either myosin or actin and our hypothesis is that the obligatory role for a reduction in Ca2+ to promote the catch state reflects the inactivation of myosin and an increase in low-force state. This complex is proposed to represent at least part of the mechanical connection (catch bridge) between the myofilaments. Serotonin release causes an increase in [cAMP] and activation of PKA. One of the PKA sites on twitchin is the D2 site and its phosphorylation eliminates binding of the D2 peptide to actin and myosin. The catch connection is lost and the muscles enters the relaxed phase.
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Alyonycheva, T. N., Mikawa, T., Reinach, F. C. and Fischman, D.
A. (1997). Isoform-specific interaction of the myosin-binding
proteins (MyBPs) with skeletal and cardiac myosin is a property of the
C-terminal immunoglobulin domain. J. Biol. Chem.
272,20866
-20872.
Andruchova, O., Hopflinger, M. C., Andruchov, O. and Galler, S. (2005). No effect of twitchin phosphorylation on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state? Eur. J. Physiol. 450,326 -334.[CrossRef][Medline]
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