Ca2+ and Cross-Bridge-Induced Changes in Troponin C in Skinned Skeletal Muscle Fibers: Effects of Force Inhibition

Abstract

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5' isomer of iodoacetamidotetramethylrhodamine (5'IATR), attached to Cys98 (sTnC-5'ATR), in sTnC-5'ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca2+ and cross-bridge binding on sTnC structure, maximum Ca2+-activated force 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. Dichroism was 0.08 +/- 0.01 (+/- SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 +/- 0.002 (+/- SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca2+ sensitivity of dichroism. Similarly, inhibition 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 normalized value at pCa 4.0 (0.11 +/- 0.05, +/- SEM; n = 3). Inhibition of force with AlF4- also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-(N-(iodoacetoxy)ethyl-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 microm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment 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; force was 0.66 +/- 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca2+].

A Mutation in TNNC1-encoded Cardiac Troponin C, TNNC1-A31S, Predisposes to Hypertrophic Cardiomyopathy and Ventricular Fibrillation

“…In comparison, the previously described HCM cTnC mutants A8V and D145E increased thin filament Ca 2ϩ affinity with (⌬pCa 50 s of ϩ0.14 and ϩ0.08), respectively (33). It is well known that the number of cross-bridges can modulate both Ca 2ϩ sensitivity and TnC affinity for the thin filament (41)(42)(43)(44)(45)(46)(47). Therefore, the addition of myosin S1 was used to assess whether strong cross-bridge formation indirectly (ϪATP) modulated the Ca 2ϩ affinity of the mutant containing thin and thick filament differently than the WT (41).…”

Cardiac troponin C (TnC) and a site I skeletal TnC mutant alter Ca2+versus crossbridge contribution to force in rabbit skeletal fibres

“…These observations are consistent with the interpretation that lowering [ATP] to 0.5 mm results in some increase in the time crossbridges spend in the rigor state (Regnier et al 1998b). Rigor crossbridges have been shown to activate thin filaments in skinned skeletal fibres through an allosteric mechanism that may increase Ca 2+ binding, as evidenced by increasing the amount of Ca 2+ bound to sTnC (Fuchs, 1985) and by inducing changes in sTnC structure (Martyn et al 1999). While slowed crossbridge detachment with low [ATP] does not increase the apparent level of thin filament activation in native skinned skeletal fibres, as evidenced by no change in pCa 50 or n H (Regnier et al 1998b), in xsTnC reconstituted fibres pCa 50 and n H were increased by low [ATP] towards control values (Fig.…”

Familial hypertrophic cardiomyopathy mutations in troponin I (K183Δ, G203S, K206Q) enhance filament sliding

“…The fluorescent probe, the 5Ј isomer of iodoacetamidotetramethylrhodamine (5Ј-IATR), was a generous gift of Dr. John Corrie (National Institute for Medical Research, Mill Hill, London). The recombinant cTnC mutant, xcTnC, was labeled with 5Ј-IATR under denaturing conditions as previously described for sTnC (41). Native bovine cTnT was similarly labeled at Cys 39 under denaturing conditions.…”