The Role of the NH2- and COOH-terminal Domains of the Inhibitory Region of Troponin I in the Regulation of Skeletal Muscle Contraction

Abstract

The role of the inhibitory region of troponin (Tn) I in the regulation of skeletal muscle contraction was studied with three deletion mutants of its inhibitory region: 1) complete (TnI-(⌬96 -116)), 2) the COOH-terminal domain (TnI-(⌬105-115)), and 3) the NH 2 -terminal domain (TnI-(⌬95-106)). Measurements of Ca 2؉ -regulated force and relaxation were performed in skinned skeletal muscle fibers whose endogenous TnI (along with TnT and TnC) was displaced with high concentrations of added troponin T. Reconstitution of the Tn-displaced fibers with a TnI⅐TnC complex restored the Ca 2؉ sensitivity of force; however, the levels of relaxation and force development varied. Relaxation of the fibers (pCa 8) was drastically impaired with two of the inhibitory region deletion mutants, TnI-(⌬96 -116)⅐TnC and TnI-(⌬105-115)⅐TnC. The TnI-(⌬95-106)⅐TnC mutant retained ϳ55% relaxation when reconstituted in the Tn-displaced fibers. Activation in skinned skeletal muscle fibers was enhanced with all TnI mutants compared with wild-type TnI. Interestingly, all three mutants of TnI increased the Ca 2؉ sensitivity of contraction. None of the TnI deletion mutants, when reconstituted into Tn, could inhibit actin-tropomyosin-activated myosin ATPase in the absence of Ca 2؉ , and two of them (TnI-(⌬96 -116) and TnI-(⌬105-115)) gave significant activation in the absence of Ca 2؉ . These results suggest that the COOH terminus of the inhibitory region of TnI (residues 105-115) is much more critical for the biological activity of TnI than the NH 2 -terminal region, consisting of residues 95-106. Presumably, the COOH-terminal domain of the inhibitory region of TnI is a part of the Ca 2؉ -sensitive molecular switch during muscle contraction.Contraction of skeletal muscle is initiated by the binding of Ca 2ϩ to the regulatory sites of troponin (Tn) 1 C, the Ca 2ϩ -binding subunit of Tn, which causes an interaction with TnI, the inhibitory subunit of Tn, releasing its inhibition of actomyosin ATPase activity. For full biological function, Tn requires a third subunit, TnT, which anchors the TnI⅐TnC complex to the actin-tropomyosin (Tm) filaments and also plays a role in the Ca 2ϩ -mediated activation of actomyosin ATPase activity and/or contraction (1-5).The inhibitory function of Tn has been studied in many different ways, yet the structure-function of the inhibitory region of TnI, responsible for this, is still under investigation. The role of the putative inhibitory region of TnI, which consists of 21 amino acids (residues 96 -116), has been studied previously utilizing synthetic peptides as well as proteolytic and recombinant fragments of TnI. The cyanogen bromide fragment (CN4) of TnI (residues 96 -116) was originally found to possess all of the inhibitory properties of intact TnI (6). Studies with synthetic peptides have demonstrated that residues 105-114 represent the minimal sequence necessary to produce inhibition of actomyosin ATPase activity and to retain TnC binding (7-10); however, the CN4 fragment (TnI-(96 -116)) has been shown to have ...

Functional Analysis of a Troponin I (R145G) Mutation Associated with Familial Hypertrophic Cardiomyopathy

“…1). Also, results from our laboratory using mutants of skeletal TnI suggest that residues 105-115 of the inhibitory region (comparable to residues 137-147 of HCTnI) are much more critical for the biological activity of TnI than residues 96 -106 (39). From this standpoint, a significant amino acid change in this region could definitely affect mechanisms of cardiac muscle contraction.…”

Solution Structure of the Chicken Skeletal Muscle Troponin Complex Via Small-angle Neutron and X-ray Scattering

“…It is likely that these segments undergo structural transitions that are driven by Ca 2C binding to the regulatory head and are likely to be dependent on the structural context. NMR studies of peptides from the central inhibitory region (I p ) of skeletal TnI, which alone can inhibit acto-myosin ATPase, summarized by Szczesna et al, 29 have revealed an extended, bent structure: residues 104-115 formed an amphiphilic helix-like structure, bent around two central proline residues, 30 while the longer 96-115 sequence appeared largely non-helical with a kink at Gly104. 31 It has also been noted 32 that I p shows sequence homology to an actin-binding loop in profilin, an actin-binding protein.…”

Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity of Force Development in the Presence of Slow Skeletal Troponin I

“…However, the presence of ssTnI instead of cTnI resulted in a decrease in the cooperativity of force development that was statistically significant (p Ͻ 0.05) when cTnT4 was utilized (Table I). Although the molecular mechanism of cooperativity is still not clearly understood, the Ca 2ϩ dependence of muscle contraction is known to be highly cooperative with Hill coefficients ranging from 1 to 4 depending on the system used (33)(34)(35). Biochemical studies on fast skeletal TnT (fsTnT) peptides suggest that part of its N-terminal region (residues 70 -159, corresponding to residues 102-189 of cTnT3) promotes cooperative interactions between functional units (36,37).…”