Da. Martyn et al., Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: Effects of force inhibition, BIOPHYS J, 76(3), 1999, pp. 1480-1493
Changes in skeletal troponin C (sTnC) structure during thin filament activa
tion by Ca2+ end strongly bound cross-bridge states were monitored by measu
ring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhoda
mine (5'IATR), attached to Cys(98) (sTnC-5'ATR), in sTnC-5'ATR reconstitute
d single skinned fibers from rabbit psoas muscle. To isolate the effects of
Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated fo
rce was inhibited with 0.5 mM AlF4- or with 30 mM 2,3 butanedione-monoxime
(BDM) during measurements of the Ca2+ dependence of force and dichroism. Di
chroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) a
nd decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dich
roism had similar Ca2+ sensitivities. Force inhibition with BDM caused no c
hange in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibi
tion of force at pCa 4.0 with 0.5 mM AlF4- decreased force to 0.04 +/- 0.01
of maximum (+/- SEM, n = 3), and dichroism was 0.04 +/- 0.03 (+/- SEM, n =
3) of the value at pCa 9.2 and unchanged relative to the corresponding nor
malized Value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of for
ce with AlF4- also had no effect when sTnC structure was monitored by label
ing with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-
(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). i
ncreasing sarcomere length from 2.5 to 3.6 mu m caused force (pCa 4.0) to d
ecrease, but had no effect on dichroism. In contrast, rigor cross-bridge at
tachment caused dichroism at pCa 9.2 to decrease to 0.56 +/- 0.03 (+/- SEM,
n = 5) of the value at pCa 9.2, and force was 0.51 +/- 0.04 (+/- SEM, n =
6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to
0.19 +/- 0.03 (+/- SEM, n = 6), slightly above the pCa 4.0 control level; f
orce was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cros
s-bridge binding in the rigor state alters sTnC structure, whereas cycling
cross-bridges have little influence at either submaximum or maximum activat
ing [Ca2+].