Vol.
166,
No.
February
14,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
TRIFLUOPERAZINE
INHIBITION
SKELETAL,
OF CONTRACTION
CARDIAC
kvind
AND SMOOTH
1421-1428
IN PERMEABILIZED MUSCLES
Babu and Jagdish Gulati
Albert Einstein College of Medicine, Department of Medicine and PhysioloGy/siophysics, 1300 Morris Park Avenue, Bronx, NY 10461 Received
January
8, 1990
To gain insightsinto the mechanismof the central helix of calmodulinand troponinC in the Ca”‘-regulation of force developmentin striated and smoothmuscles,the present study was made of the TFP induced inhibition of contraction, and of the uptake of theseproteins by skinnedfibers. Calmodulinwasfour-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac musclesdespite the differences in their troponin-C isoforms. Also, the results werecomparablebetweenfast-twitch fiber, when calmodulinwasexchanged for troponin-C to act on TnI, and smoothmuscle,where calmodulin acts on myosin light chain kinase. Thesefindings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sitesin the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFPindependentdomain in troponin-C that binds Tnl. @1990 Academic Press,1°C.
Since the original observations of Ringer [l] on the dependence of heart contractions on extracellular
calcium, and of Heilbrun
and Wiercinski
[2] showing the contractile effect
of the injected ion, Ca2’ is now well established as a triggering
messenger for a large
variety of functions in eukaryotic cells. The trigger mechanism is initiated
by the binding
of Ca”
in the cell [3].
to calcium-modulated
proteins, several of which are available
Amongst these proteins, calmodulin characterized
and troponin
[4-61. Both have four Ca”-binding
C are most similar and also the best sites, in two groups of two each and
separated by an unusually long (approx. 40A) central helix. The role of the central helix in modulating
the interaction
between the two groups has been the subject of much interest
[7-111, but as yet the mechanism remains unknown. Presently, we report the studies of the inhibition of calmodulin and troponin-C by trifluoperazine (TFP) in the skinned fibers that provide new insights into the function of the central helix in the contraction-relaxation switching mechanism. The abbreviations used are; CaM, calmodulin; TnC, troponin-C; TnI, troponin I; TnT, troponin T, TM, tropomyosin; MLC’s, myosin light chains; MLCK, kinase; TFP, trifluoperazine; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol bis (l3aminoethyl ether)-NJ’-tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
1421
0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproductiorz in any form reserved.
Vol.
166, No. 3, 1990
BIOCHEMICAL
TFP is a well known phenothiazine
AND BIOPHYSICAL
inhibitor
RESEARCH COMMUNICATIONS
of the calmodulin
function [12,13]. It
also inhibits the force development in muscle [14,15]. TFP acts by binding two hydrophobic sites within the central helix of calmodulin and troponin-C [16]. These hydrophobic sites are exposed in the presence of calcium. Here we demonstrate
for the first time that the
uptake of calmodulin in exchange for troponin-C by the striated muscle fibers was blocked with TFP. The results suggest that the modified conformation of the central-helix in the TFP-calmodulin
complex is incompatible
with the uptake sites in the fiber, and/or that the
binding of TFP to the central helix screens the attachment site on the modulator. other hand, the results with troponin-C this case, while blocking modulator,
show that the conformational
On the
change with TFP in
the force, causes no hinderance to the uptake or release of the
which indicates further that troponin-C
contains an additional
TFP-insensitive
domain that functions for binding in the fiber. In addition, by comparing the TFP response on permeabilized
skeletal and smooth
muscles, we also suggest that there are common key steps in the process of the interaction of calmodulin
with various target proteins, such as TnI and myosin light chain kinase. The
execution of these key steps, for the contraction-relaxation switch in different tissues, appears to be modulated similarly by the activity of the hydrophobic domains in the central helix. METHODS
Fiber preparation & skinning. Three types of muscle preparations were used in this study: fast-twitch fibers of rabbit psoas muscle, thin trabeculae for cardiac muscle samples from the right ventricle of the hamster, and taenia coli for smooth muscle samples from the guinea-pig. For fast-twitch fibers, small bundles (l-2mm by 4-10mm) of psoas muscle of adult rabbit were tied to sticks and stored overnight at -2O’C for skinning in solution containing 150mM potassium propionate, 5mM Mg-acetate, 5mM EGTA, 5mM ATP, 1mM dithiothreiotol, 50% v/v glycerol, and pH = 7.00, as described earlier [17,18]. Single fiber segment was isolated and attached to the shaft of a force transducer, and transferred to relaxing solution (100mM potassium propionate, 20mM imidazole, 6.06mM MgCl,, 5mM ATP, 5mM EGTA, 20mM phosphocreatine, pH 7.00 and ionic strength 190-200mM; 1mM free M$‘). In each case, the fiber was briefly treated with 0.5% triton-x detergent (2 min treatment at 1O’C). Activating solutions were made by appropriately varying the Ca”EGTA to EGTA ratio. Experiments were carried out at the sarcomere length of 2.5pm as monitored by laser diffraction. Activations were made at 5’C and 20°C, as indicated. For cardiac muscle, trabeculae 80-150pm (width) by l-3mm (length) were dissected and treated with 0.5% lubrol-WX detergent in the relaxing solution for 10 min at 10°C for skinning. T’he sarcomere length was adjusted at 2.2Fm during the experiment. For smooth muscle, samples of taenia coli were isolated from the freshly-sacrificed animal [19], and transferred to a skinning solution exactly as above for psoas muscle. The experimental 1422
Vol.
166,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
preparation was a carefully dissected strip about ZOOpm x 2-3mm, and the length of the strip was adjusted to remove slack at zero resting tension.
Troponin-C
extraction from the fiber and reconstitution.
This protocol was applied only to the fast-twitch fibers. To extract the troponin-C, the attached fiber (2.5pm sarcomere length) was equilibrated in a Ca’+-free rigor solution (165mM potassium propionate, 20mM imidazole, 2.5mM EGTA, 2SmM EDTA, pH 7.0) at 4’C, and transferred to the extracting solution (5mM EDTA, 1OmM imidazole, pH 7.2) at 30°C for 5 to 30 min duration as described earlier [ 17,181. Force with maximal activation (pCa4) was checked periodically, and extraction was ended when force was below 2O%P. SDS gel runs on such fibers indicated a 70-75% troponin-C extraction. To recombine troponin-C with denuded sites in the fiber, 10 to 30min incubation at 5’C was given (0.2 to lmg/ml protein in the relaxing solution). Afterwards the free protein was washed out with several rinses, unless otherwise indicated. The insertion of calmodulin (commercially purified from bovine brain; Sigma) was made by loading the fibers in the activating solution at the concentration of 200pg/ml [17]. Rinsing was necessary to run the gels, and was carried out also in the activating solution. All experimental fibers were stored at -7O’C for quantitative analysis by gel electrophnresis. The stored fibers were ultrasonicated in the sample buffer containing 1mM EGTA for gel loading. The gels were silver-stained for resolving the troponin-C and calmodulin bands in single fiber segments. The running of gels and staining were also similar to those described earlier [ 17,181. The intensities of the various protein bands were quantitatively analyzed with a laser densitometer of 100pm beam resolution. The statistics of the data are given as +S.E.M.
wherever appropriate.
RESULTS
Effect of TFP on calmodulin- and TnC-regulated psoas fibers. Fig.1 shows the force traces on a fiber to indicate the uptake of calmodulin presence of Ca” (pCa4). The first trace in a shows the contraction-relaxation native fiber (force: PO). After troponin-C-extraction, the force was diminished. in B shows the response with commercially
purified brain calmodulin
in the
cycle on the The last trace
(200pg/ml)
in the
activating solution. The force rises slowly, reaching the plateau (0.71Po) in about lo-llmin. The plateau was quite well maintained for several minutes even after transferring the fiber to a calmodulin-free
solution; the force in calmodulin-free
pCa4 solution eventually reached
zero level after 4 to 6 hours, indicting that the release of Ca”-calmodulin complex is slow on this time scale. However, including 100pM TFP in the activating solution dramatically accelerated the drop in force (trace b). Further, if the same maximal concentration of TFP was included in the calmodulin-loading solution, force never increased above the zero level 1423
Vol.
BIOCHEMICAL
166, No. 3, 1990 a
100 kNhn’ i
;I
natike
A I1
i%
RESEARCH COMMUNICATIONS
t
EaM - pCa 4
-itiT
b
AND BIOPHYSICAL
pCa 4 (0 CaM)
40s
A Eat+ pCa 4
fFP- pCa 4
laM-TFP-
pCa 4
F&& Force responsewith calmodulin. (a) The first trace is the force responseof the unextracted fiber to pCa4 activation. Force wasdiminishedafter the fiber wasextracted in the secondtrace. Transferring the fiber into an activating solution with calmodulin also gaveforce but at a slowertime scale.The interruptions in the force trace with calmodulin are due to imposedrelease-stretchlength stepsduring prolonged activations.The force decreasedslowly on the removal of free calmodulinfrom the solution. (b) The drop in force was acceleratedwith TFP. (c) Including TFP initially completelyblocked the force. All activations at SC.
(trace s). These observations suggest that TFP both blocks the uptake of calmodulin
fiber as well as accelerates the release of bound calmodulin
in the presence of Ca’“.
Fig.2 shows the full dose-response curve of TFP on calmodulin-loaded shown is the effect on native (skinned, unextracted)
by the
fibers. Also
psoas fibers as well as on cardiac
muscle. These fibers containing
troponin-C
are found to be 1/4th to 1/5th as sensitive to
TFP as the calmodulin-loaded
fibers. To rule out that this difference was due to the
cardiac
m. - A
0” .E a 2 E
0.5-
.-skeletal q -smooth
m. (+c&) m
z
10
TFP
20
50
100
concentration
(PM)
200
500
&&. The inhibition of force by TFP in ditTerentfibers.The temperatureduring activations wasS°Cfor psoasfibers, and 20°Cfor cardiacand smoothmuscles.The effects on psoas fibers were checkedat 20°Candwere similar,except that the fiber wasmore stableat the lower temperature. 1424
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Results 1) Expressionof calpastatinsegments in E. coli Calpastatinsegments were first producedin E. coli harboringthe expressionplasmidswe constructedandsix of them,~42, ~421,~422, ~94, ~65, and~81 werethen purified. These segments weremainly composedof domain1 of humancalpastatinbut they weretruncatedat different positionsat eitherthe aminoterminalor the carboxy terminalside(Fig. 2). Four of them(~42, ~421, ~422, and~94) coveredthe N-sequence,but the other two (p65 and~81) did not. ~421and~422 hadthe sameaminoterminusasp42 but weretruncatedat different positionsat the carboxy terminalside; ~421 hadthe C-sequence, but ~422 did not.
.. . . ..
70-w--
422
,
-3s
1
,.*.4..*
-m
31p
(0)”
‘9
CC)
K,.,"S,F $“A:
M *;
A
A
L
D
D
L
I
4
T
L.
G
G
370
94
GGTGACAAGAAAAAAGAAAAGAA;LTCATTAACICCCAGCTGTGC~AGTTGAATCTAAACCGGATiAACCATCGGGA SLTPAVPVEfjKPDKPSG
P
E
E
T5$
5
El;
4
S
GyTATTRCToCGA~CAgAAGT~~CADDAT~CA~TG~GT~CC~CC~ACATAGAGGAATTGGGTAAAAGAGAA~TC 1EELg;KUE . 16b
935 .
ACA:TTCCTCCAAAATATAGGGAiCTATTGGCT T YRELLA
995
k&I
CCCATAGdGCCAGATGATGCTATAGAC&CTTGTCATCT G P D DA I D A
610 . GACTTCiYC D F 665
i&K
~GG~CGCCTACAGCTGCTGGAAAGAAAACTGAA~AA~AGGAA~CTACAGAA~TT~TA~AA~CT P2$*AAGKKTE' 5:
?sa
CAG'+CAGCAGGGACAGTCAGAAG;GCTGCTCCACCCCAAGAGAiG QSqGTVRSAAPP
p
E
K
#Jf;GA;AG~TG~AG~AG !uA
1488
TS;
S
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
that TFP readily dissociates all of the calmodulin
RESEARCH COMMUNICATIONS
in the fiber. Because calmodulin
was not
present in the TFP solution, a number of additional controls were performed to check this point.: We got the same results on SDS-PAGE as above: 1) if calmodulin was included with TFP in the Ca” solution. The fall in force from the plateau was also similar to that in the solution with TFP alone (see Fig.lb). And, 2) if the fiber was taken directly to a TFPcalmodulin solution, such that there was no rise in force at all (see Fig.lc). In both cases, no calmodulin
was found to be retained in the fiber.
We next used TFP with the native fiber to see the effect on bound troponin-C. After exposure to TFP to block the force (500pM; Fig 2), the fiber was rinsed free of TFP and reactivated
(Fig.4A).
Force was normal,
unaltered. A TnC-extracted
indicating
that the troponin-C
fiber was also loaded with troponin-C
and the uptake was normal indicate that the inhibition
content was
in the presence of TFP
as checked by the force response (Fig.4B). These findings of force by TFP is a direct effect of the conformational
induced by TFP in the central helix, but that there is also a TFP-insensitive
change domain in
troponin-C
regulating the binding of the modulator in the fiber. Our results suggest that this
additional
domain is absent in calmodulin. DISCUSSION
This is the first study simultaneously the contraction-relaxation
comparing the inhibitory
switches operated by calmodulin
properties of TFP on
and troponin-C
the different muscle types. On psoas muscle, we find that the inhibition was associated with blocked uptake of calmodulin, indicate that the troponin-C
in the fibers of of force by TFP
but not of troponin-C.
interaction with TnI includes an additional
The results
domain, not present
in calmodulin and, therefore, probably, also not directly involved in the regulatory function. The additional interaction of troponin-C to TnI is presumably important to muscle, in the evolutionary sense, as it would facilitate attachment of the modulator to its target site, and help its retention, for more efficient contraction. Because the global structures of troponin-C inhibition
of the regulatory
and calmodulin
functions by TFP, very likely
are so similar,
the
is the result of a similar
conformational change of the modulators (calmodulin and troponin-C) with the active sites on the target protein. In vitro biochemical studies have indicated that the inhibitory action of TFP is associated with its binding to two hydrophobic domains in the central helix [16]. Further, because the action of TFP on cardiac and skeletal isoforms of troponin-Cs is now shown to be identical, despite important differences in the Ca”-binding properties of these proteins [21,22], the fiber results also provide additional evidence that TFP acts entirely through the central helix of the modulators. Furthermore, the comparison of the results of TFP inhibition of calmodulin between skeletal and smooth muscle strips provides insights into whether the hydrophobic domain 1426
Vol.
166, No. 3, 1990
BIOCHEMICAL
in the central helix have similar target proteins. The inhibition the Ca”-switch
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
or separate mechanisms in the regulations
of the smooth muscle strips, utilizing
of different
MLCK-calmodulin
as
[20], was found to be similar to that of striated fibers where calmodulin
was
acting on TnI. These results support the first possibility above, showing that at least some of the steps overlap between the interactions of calmodulin with TnI and MLCK. In conclusion, the present findings show that the hydrophobic domains in the central helix of calmodulin and troponin-C perform a common critical function in the contractionrelaxation switching mechanisms in both striated and smooth muscles. Further, the binding of TFP to the central helix appears to inactivate the same key step in the interaction modulator
of the
with target proteins as varied as myosin light chain kinase in smooth muscle and
TnI in striated muscle. ACKNOWLEDGMENTS The work was supported by NIH grant AR-33736, and a New York Heart Grant-in-aid. REFERENCES
PI-
Ringer, S. (1883) J. Physiol. London 4, 29-42.
M.
Heilbrun,
[31*
Cox, J.A., Comte, M., Malone, A., Burger, D., and Stein, E.A. (1984) In Metal Ions
L.V., and Wiercinski,
J. (1947) J. Cell. camp. Physiol. 29, 15-32.
in Biological Systems(H.Sigel,Ed.),Vol.17,pp.215-273.
Marcel Dekker, New York.
141.
Babu, Y.S. et al (1985) Nature 31537-42.
PI* [61-
Herzberg, O., and James, M.N. (1985) Nature 313,653-659. Sundralingam, M. et al (1985) Science 227, 945-948.
171.
Putkey, J.A., Ono, T., VanBerkum,
PI
263, 11242-l 1249. Craig, T.A., Watterson,
M.F.A., and Means, A.R. (1988) J. Biol. Chem.
D.M., Prendergast, F.G., Haiech, J., and Roberts, D.M.
(1987) J. Biol. Chem. 262, 3278-3284. [91*
Persechini, A et al (1989) J. Biol. Chem. 264, 8052-8058.
WI. Xu, G., and Hitchcock, S.E. (1988) J. Biol. Chem. 263, 13962-13969. [111* Reinach, F.C., and Karlsson, R. (1988) J. Biol. Chem. 263, 2371-2376. WI*
Stoclet, J. et al (1987) Prog. Neurology 29, 321-364.
[131.
Weiss, B. et al (1985) In Calmodulin Antagonists & Cellular Physiology (H.Hidaka D.Hartshorne, Eds.),pp.45-61. Academic Press, NY.
PI-
Kurebayashi, N., and Ogawa, Y. (1988) J. Physiol London 403, 407-424.
1151. Moreland,
S., Little,
D.K., and Moreland,
&
R.S. (1987) Am. J. Physiol. 252, C523-
c531.
[161. Strynadka, N.C.J., and James, N.G. (1988) Proteins: Strut. Func. Genetics 3, 1-17. P71-
Babu, A., Orr, G., and Gulati, J. (1988) J. Biol. Chem. 263, 15485-15491. 1427
Vol.
[18].
166, No. 3, 1990
BIOCHEMICAL
Babu, A., Scordilis, S., Sonnenblick,
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
E., and Gulati,
J. (1987) J. Biol. Chem. 262,
5815-5822. [19].
Reisin, I.L., and Gulati, J. (1972) Science 176, 170-177.
[20].
Hartshorne,
D.J. (1987) In Physiology of the Gastrointestinal Tract (L.R. Johnson,
Ed.), pp.423-482. Raven Press, New York
WI.
vanEerd, J.P., and Takahashi,
K. (1975) Biochem. Biophys. Res. Commun.64, 122-
127. [22].
Gulati, J., Babu, A., and Putkey, J.A. (1989) Febs Lett. 248, 5-8.
1428
166,
No.
February
14,
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
TRIFLUOPERAZINE
INHIBITION
SKELETAL,
OF CONTRACTION
CARDIAC
kvind
AND SMOOTH
1421-1428
IN PERMEABILIZED MUSCLES
Babu and Jagdish Gulati
Albert Einstein College of Medicine, Department of Medicine and PhysioloGy/siophysics, 1300 Morris Park Avenue, Bronx, NY 10461 Received
January
8, 1990
To gain insightsinto the mechanismof the central helix of calmodulinand troponinC in the Ca”‘-regulation of force developmentin striated and smoothmuscles,the present study was made of the TFP induced inhibition of contraction, and of the uptake of theseproteins by skinnedfibers. Calmodulinwasfour-fold more sensitive to TFP than TnC, but the inhibition was found to be identical for skeletal and cardiac musclesdespite the differences in their troponin-C isoforms. Also, the results werecomparablebetweenfast-twitch fiber, when calmodulinwasexchanged for troponin-C to act on TnI, and smoothmuscle,where calmodulin acts on myosin light chain kinase. Thesefindings indicate that the inhibition of force by TFP is entirely due to its binding to the hydrophobic sitesin the central helix. The uptakes of troponin-C and calmodulin were also different, and this is explained by a TFPindependentdomain in troponin-C that binds Tnl. @1990 Academic Press,1°C.
Since the original observations of Ringer [l] on the dependence of heart contractions on extracellular
calcium, and of Heilbrun
and Wiercinski
[2] showing the contractile effect
of the injected ion, Ca2’ is now well established as a triggering
messenger for a large
variety of functions in eukaryotic cells. The trigger mechanism is initiated
by the binding
of Ca”
in the cell [3].
to calcium-modulated
proteins, several of which are available
Amongst these proteins, calmodulin characterized
and troponin
[4-61. Both have four Ca”-binding
C are most similar and also the best sites, in two groups of two each and
separated by an unusually long (approx. 40A) central helix. The role of the central helix in modulating
the interaction
between the two groups has been the subject of much interest
[7-111, but as yet the mechanism remains unknown. Presently, we report the studies of the inhibition of calmodulin and troponin-C by trifluoperazine (TFP) in the skinned fibers that provide new insights into the function of the central helix in the contraction-relaxation switching mechanism. The abbreviations used are; CaM, calmodulin; TnC, troponin-C; TnI, troponin I; TnT, troponin T, TM, tropomyosin; MLC’s, myosin light chains; MLCK, kinase; TFP, trifluoperazine; EDTA, ethylene diamine tetraacetic acid; EGTA, ethylene glycol bis (l3aminoethyl ether)-NJ’-tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.
1421
0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproductiorz in any form reserved.
Vol.
166, No. 3, 1990
BIOCHEMICAL
TFP is a well known phenothiazine
AND BIOPHYSICAL
inhibitor
RESEARCH COMMUNICATIONS
of the calmodulin
function [12,13]. It
also inhibits the force development in muscle [14,15]. TFP acts by binding two hydrophobic sites within the central helix of calmodulin and troponin-C [16]. These hydrophobic sites are exposed in the presence of calcium. Here we demonstrate
for the first time that the
uptake of calmodulin in exchange for troponin-C by the striated muscle fibers was blocked with TFP. The results suggest that the modified conformation of the central-helix in the TFP-calmodulin
complex is incompatible
with the uptake sites in the fiber, and/or that the
binding of TFP to the central helix screens the attachment site on the modulator. other hand, the results with troponin-C this case, while blocking modulator,
show that the conformational
On the
change with TFP in
the force, causes no hinderance to the uptake or release of the
which indicates further that troponin-C
contains an additional
TFP-insensitive
domain that functions for binding in the fiber. In addition, by comparing the TFP response on permeabilized
skeletal and smooth
muscles, we also suggest that there are common key steps in the process of the interaction of calmodulin
with various target proteins, such as TnI and myosin light chain kinase. The
execution of these key steps, for the contraction-relaxation switch in different tissues, appears to be modulated similarly by the activity of the hydrophobic domains in the central helix. METHODS
Fiber preparation & skinning. Three types of muscle preparations were used in this study: fast-twitch fibers of rabbit psoas muscle, thin trabeculae for cardiac muscle samples from the right ventricle of the hamster, and taenia coli for smooth muscle samples from the guinea-pig. For fast-twitch fibers, small bundles (l-2mm by 4-10mm) of psoas muscle of adult rabbit were tied to sticks and stored overnight at -2O’C for skinning in solution containing 150mM potassium propionate, 5mM Mg-acetate, 5mM EGTA, 5mM ATP, 1mM dithiothreiotol, 50% v/v glycerol, and pH = 7.00, as described earlier [17,18]. Single fiber segment was isolated and attached to the shaft of a force transducer, and transferred to relaxing solution (100mM potassium propionate, 20mM imidazole, 6.06mM MgCl,, 5mM ATP, 5mM EGTA, 20mM phosphocreatine, pH 7.00 and ionic strength 190-200mM; 1mM free M$‘). In each case, the fiber was briefly treated with 0.5% triton-x detergent (2 min treatment at 1O’C). Activating solutions were made by appropriately varying the Ca”EGTA to EGTA ratio. Experiments were carried out at the sarcomere length of 2.5pm as monitored by laser diffraction. Activations were made at 5’C and 20°C, as indicated. For cardiac muscle, trabeculae 80-150pm (width) by l-3mm (length) were dissected and treated with 0.5% lubrol-WX detergent in the relaxing solution for 10 min at 10°C for skinning. T’he sarcomere length was adjusted at 2.2Fm during the experiment. For smooth muscle, samples of taenia coli were isolated from the freshly-sacrificed animal [19], and transferred to a skinning solution exactly as above for psoas muscle. The experimental 1422
Vol.
166,
No.
3, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
preparation was a carefully dissected strip about ZOOpm x 2-3mm, and the length of the strip was adjusted to remove slack at zero resting tension.
Troponin-C
extraction from the fiber and reconstitution.
This protocol was applied only to the fast-twitch fibers. To extract the troponin-C, the attached fiber (2.5pm sarcomere length) was equilibrated in a Ca’+-free rigor solution (165mM potassium propionate, 20mM imidazole, 2.5mM EGTA, 2SmM EDTA, pH 7.0) at 4’C, and transferred to the extracting solution (5mM EDTA, 1OmM imidazole, pH 7.2) at 30°C for 5 to 30 min duration as described earlier [ 17,181. Force with maximal activation (pCa4) was checked periodically, and extraction was ended when force was below 2O%P. SDS gel runs on such fibers indicated a 70-75% troponin-C extraction. To recombine troponin-C with denuded sites in the fiber, 10 to 30min incubation at 5’C was given (0.2 to lmg/ml protein in the relaxing solution). Afterwards the free protein was washed out with several rinses, unless otherwise indicated. The insertion of calmodulin (commercially purified from bovine brain; Sigma) was made by loading the fibers in the activating solution at the concentration of 200pg/ml [17]. Rinsing was necessary to run the gels, and was carried out also in the activating solution. All experimental fibers were stored at -7O’C for quantitative analysis by gel electrophnresis. The stored fibers were ultrasonicated in the sample buffer containing 1mM EGTA for gel loading. The gels were silver-stained for resolving the troponin-C and calmodulin bands in single fiber segments. The running of gels and staining were also similar to those described earlier [ 17,181. The intensities of the various protein bands were quantitatively analyzed with a laser densitometer of 100pm beam resolution. The statistics of the data are given as +S.E.M.
wherever appropriate.
RESULTS
Effect of TFP on calmodulin- and TnC-regulated psoas fibers. Fig.1 shows the force traces on a fiber to indicate the uptake of calmodulin presence of Ca” (pCa4). The first trace in a shows the contraction-relaxation native fiber (force: PO). After troponin-C-extraction, the force was diminished. in B shows the response with commercially
purified brain calmodulin
in the
cycle on the The last trace
(200pg/ml)
in the
activating solution. The force rises slowly, reaching the plateau (0.71Po) in about lo-llmin. The plateau was quite well maintained for several minutes even after transferring the fiber to a calmodulin-free
solution; the force in calmodulin-free
pCa4 solution eventually reached
zero level after 4 to 6 hours, indicting that the release of Ca”-calmodulin complex is slow on this time scale. However, including 100pM TFP in the activating solution dramatically accelerated the drop in force (trace b). Further, if the same maximal concentration of TFP was included in the calmodulin-loading solution, force never increased above the zero level 1423
Vol.
BIOCHEMICAL
166, No. 3, 1990 a
100 kNhn’ i
;I
natike
A I1
i%
RESEARCH COMMUNICATIONS
t
EaM - pCa 4
-itiT
b
AND BIOPHYSICAL
pCa 4 (0 CaM)
40s
A Eat+ pCa 4
fFP- pCa 4
laM-TFP-
pCa 4
F&& Force responsewith calmodulin. (a) The first trace is the force responseof the unextracted fiber to pCa4 activation. Force wasdiminishedafter the fiber wasextracted in the secondtrace. Transferring the fiber into an activating solution with calmodulin also gaveforce but at a slowertime scale.The interruptions in the force trace with calmodulin are due to imposedrelease-stretchlength stepsduring prolonged activations.The force decreasedslowly on the removal of free calmodulinfrom the solution. (b) The drop in force was acceleratedwith TFP. (c) Including TFP initially completelyblocked the force. All activations at SC.
(trace s). These observations suggest that TFP both blocks the uptake of calmodulin
fiber as well as accelerates the release of bound calmodulin
in the presence of Ca’“.
Fig.2 shows the full dose-response curve of TFP on calmodulin-loaded shown is the effect on native (skinned, unextracted)
by the
fibers. Also
psoas fibers as well as on cardiac
muscle. These fibers containing
troponin-C
are found to be 1/4th to 1/5th as sensitive to
TFP as the calmodulin-loaded
fibers. To rule out that this difference was due to the
cardiac
m. - A
0” .E a 2 E
0.5-
.-skeletal q -smooth
m. (+c&) m
z
10
TFP
20
50
100
concentration
(PM)
200
500
&&. The inhibition of force by TFP in ditTerentfibers.The temperatureduring activations wasS°Cfor psoasfibers, and 20°Cfor cardiacand smoothmuscles.The effects on psoas fibers were checkedat 20°Candwere similar,except that the fiber wasmore stableat the lower temperature. 1424
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166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Results 1) Expressionof calpastatinsegments in E. coli Calpastatinsegments were first producedin E. coli harboringthe expressionplasmidswe constructedandsix of them,~42, ~421,~422, ~94, ~65, and~81 werethen purified. These segments weremainly composedof domain1 of humancalpastatinbut they weretruncatedat different positionsat eitherthe aminoterminalor the carboxy terminalside(Fig. 2). Four of them(~42, ~421, ~422, and~94) coveredthe N-sequence,but the other two (p65 and~81) did not. ~421and~422 hadthe sameaminoterminusasp42 but weretruncatedat different positionsat the carboxy terminalside; ~421 hadthe C-sequence, but ~422 did not.
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935 .
ACA:TTCCTCCAAAATATAGGGAiCTATTGGCT T YRELLA
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1488
TS;
S
Vol.
166, No. 3, 1990
BIOCHEMICAL
AND BIOPHYSICAL
that TFP readily dissociates all of the calmodulin
RESEARCH COMMUNICATIONS
in the fiber. Because calmodulin
was not
present in the TFP solution, a number of additional controls were performed to check this point.: We got the same results on SDS-PAGE as above: 1) if calmodulin was included with TFP in the Ca” solution. The fall in force from the plateau was also similar to that in the solution with TFP alone (see Fig.lb). And, 2) if the fiber was taken directly to a TFPcalmodulin solution, such that there was no rise in force at all (see Fig.lc). In both cases, no calmodulin
was found to be retained in the fiber.
We next used TFP with the native fiber to see the effect on bound troponin-C. After exposure to TFP to block the force (500pM; Fig 2), the fiber was rinsed free of TFP and reactivated
(Fig.4A).
Force was normal,
unaltered. A TnC-extracted
indicating
that the troponin-C
fiber was also loaded with troponin-C
and the uptake was normal indicate that the inhibition
content was
in the presence of TFP
as checked by the force response (Fig.4B). These findings of force by TFP is a direct effect of the conformational
induced by TFP in the central helix, but that there is also a TFP-insensitive
change domain in
troponin-C
regulating the binding of the modulator in the fiber. Our results suggest that this
additional
domain is absent in calmodulin. DISCUSSION
This is the first study simultaneously the contraction-relaxation
comparing the inhibitory
switches operated by calmodulin
properties of TFP on
and troponin-C
the different muscle types. On psoas muscle, we find that the inhibition was associated with blocked uptake of calmodulin, indicate that the troponin-C
in the fibers of of force by TFP
but not of troponin-C.
interaction with TnI includes an additional
The results
domain, not present
in calmodulin and, therefore, probably, also not directly involved in the regulatory function. The additional interaction of troponin-C to TnI is presumably important to muscle, in the evolutionary sense, as it would facilitate attachment of the modulator to its target site, and help its retention, for more efficient contraction. Because the global structures of troponin-C inhibition
of the regulatory
and calmodulin
functions by TFP, very likely
are so similar,
the
is the result of a similar
conformational change of the modulators (calmodulin and troponin-C) with the active sites on the target protein. In vitro biochemical studies have indicated that the inhibitory action of TFP is associated with its binding to two hydrophobic domains in the central helix [16]. Further, because the action of TFP on cardiac and skeletal isoforms of troponin-Cs is now shown to be identical, despite important differences in the Ca”-binding properties of these proteins [21,22], the fiber results also provide additional evidence that TFP acts entirely through the central helix of the modulators. Furthermore, the comparison of the results of TFP inhibition of calmodulin between skeletal and smooth muscle strips provides insights into whether the hydrophobic domain 1426
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166, No. 3, 1990
BIOCHEMICAL
in the central helix have similar target proteins. The inhibition the Ca”-switch
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
or separate mechanisms in the regulations
of the smooth muscle strips, utilizing
of different
MLCK-calmodulin
as
[20], was found to be similar to that of striated fibers where calmodulin
was
acting on TnI. These results support the first possibility above, showing that at least some of the steps overlap between the interactions of calmodulin with TnI and MLCK. In conclusion, the present findings show that the hydrophobic domains in the central helix of calmodulin and troponin-C perform a common critical function in the contractionrelaxation switching mechanisms in both striated and smooth muscles. Further, the binding of TFP to the central helix appears to inactivate the same key step in the interaction modulator
of the
with target proteins as varied as myosin light chain kinase in smooth muscle and
TnI in striated muscle. ACKNOWLEDGMENTS The work was supported by NIH grant AR-33736, and a New York Heart Grant-in-aid. REFERENCES
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