Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C

Abstract

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.

Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride, a slowly dissociating analogue of inorganic phosphate, in chemically skinned fibres from rabbit psoas muscle

“…As has been found for Pi, the dissociation rate of tightly-binding Pi analogues from crossbridges is greatly accelerated by actin (Werber et al, 1992;Phan et al, 1993), and thus shows an apparent dependence on Ca'+ in muscle fibres (Chase 8s Kushmerick, 1990;Chase et al, 1993). In the absence of Ca2+ (or crossbridge cycling) and at physiological [MgATP], the dissociation of aluminiofluoride (AlF) or orthovanadate (Vi) from myosin in fibres is sufficiently slow (t,,, >> 15 min) as to appear to be 'trapped (Dantzig & Goldman, 1985;Chase & Kushmerick, 1990;Chase et al, 1993), although during maximum Ca'+-activation analogue dissociation became faster and t,,, decreased to 2-4 min for AlF or < 1 min for Vi (Chase & Kushmerick, 1990;Chase et al, 1993).…”

“…Tightly binding analogues of Pi also inhibit force to a greater extent than stiffness, and thus when bound to myosin, may provide a crossbridge intermediate which is analogous to the Pi-bound state occurring early in the crossbridge cycle (Chase et al, 1993). Such analogues may be particularly useful for investigations of the initial steps in the actomyosin interaction leading to force generation since dissociation of the analogue from myosin apparently occurs on a time scale which is orders of magnitude slower than Pi (Phan & Reisler, 1992;Werber et al, 1992;Chase et al, 1993).…”

“…In the absence of Ca2+ (or crossbridge cycling) and at physiological [MgATP], the dissociation of aluminiofluoride (AlF) or orthovanadate (Vi) from myosin in fibres is sufficiently slow (t,,, >> 15 min) as to appear to be 'trapped (Dantzig & Goldman, 1985;Chase & Kushmerick, 1990;Chase et al, 1993), although during maximum Ca'+-activation analogue dissociation became faster and t,,, decreased to 2-4 min for AlF or < 1 min for Vi (Chase & Kushmerick, 1990;Chase et al, 1993). There does not appear to be an absolute requirement for Ca'+, since activation by rigor crossbridges at low [MgATP] increased the Vi dissociation rate in fibres without addition of Ca'+ (Dantzig & Goldman, 1985).…”

“…Furthermore, Ca'+ may modulate Pi release kinetics and thus the steady state level of force either by binding to troponin C (TnC) alone or to additional sites such as myosin light chain 2 (MLC,) (Metzger & Moss, 1991. To address this latter possibility, we examined inhibition and recovery from inhibition of force at pCa 9.2 utilizing an activating TnC (aTnC), a structurally modified form of cardiac TnC (cTnC) which, when substituted for endogenous skeletal TnC (sTnC) in skinned skeletal muscle fibres, enables force generation and shortening independent of [Ca"'] (Hannon et al, 1993;Putkey et al, 1993).…”

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

“…Reconstitution of Tn complexes with cTnC or xsTnC was achieved by 2 or 3 min incubations in 1 mg ml −1 (total) TnC in pCa 9.2 without CK or Dextran (Hannon et al 1993;Regnier et al 1999). Reconstitution was considered complete when maximal force (rF max ) no longer increased with subsequent incubations, which occurred within 4-9 min of incubation ( Fig.…”