Ca2+ and cross-bridge-induced changes in troponin C in skinned skeletal muscle fibers: Effects of force inhibition

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+].