Abstract
Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca2+ dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca2+ dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin CIAEDANS, reported Ca2+ dissociation exclusively from the structural domain of troponin C at ∼0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca2+ dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin reported Ca2+ dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin reported a biphasic fluorescence change upon Ca2+ dissociation from the N- and C-terminal domains of troponin C with rates that were similar to those reported by troponin and troponin CIAEDANS at all levels of the troponin C systems. Furthermore, the rate of Ca2+ dissociation from troponin C in the myofibrils was similar to the rate of Ca2+ dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca2+ dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca2+ dissociation from troponin C may influence relaxation.
Highlights
Dimethylaminonaphthalene-1-sulfonyl aziridine; TnCEndog, endogenous troponin C (TnC); TnCF29W, TnC containing the F29W mutation; TnCF29W(5OH), TnCF29W containing the Trp analog 5-fluoro-dl-tryptophan; TnCIAEDANS, TnC labeled at Cys 101 with IAEDANS; TnCTIA5A4CN,SC101S, TnC containing the T54C and C101S mutations labeled on T54C with IAANS; TnCTM5I4ACN,CS101S, TnC containing the T54C and C101S mutations labeled on T54C with MIANS; TnI, troponin I; TnI96–148, chicken skeletal troponin I peptide corresponding to residues 96–148; TnIAANS, Tn complex containing TnI labeled with IAANS
We hypothesized that it www.frontiersin.org would be possible to take advantage of the fluorescence signal of TnCIAEDANS to follow the rate of Ca2+ dissociation from the C-terminal structural domain of TnC in myofibrils
As Ca2+ dissociated from isolated TnCIAEDANS, the fluorescent signal decayed at a rate of 0.37 ± 0.03/s at 15 ̊C (Figure 1A), which was similar to the rate of Ca2+ dissociation from the C-terminal sites of TnCF29W (0.48 ± 0.01/s) as measured by the fluorescent Ca2+ chelator Quin-2 (Davis et al, 2002)
Summary
Fast-twitch skeletal muscle troponin C (TnC) contains two Nterminal and two C-terminal EF-hand Ca2+ binding sites that can be distinguished according to their Ca2+ dissociation ratesbutanedione monoxime; BTS, N -benzyl-p-toluenesulfonamide; [Ca2+]50, [Ca2+] required for 50% of maximal force generation; DTT, dithiothreitol; EDTA, ethylenediamine-N,N,N ,N -tetraacetic acid; EGTA, ethylene glycol bis ((-aminoethyl ether)-N,N,N ,N -tetraacetic acid; IAANS, 2-(4 -(iodoacetamido)anilino) naphthalene-6-sulfonic acid; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid; IANBD, N,N -dimethyl-N -(iodoacetyl)-N -(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine; Kd(Ca), Ca2+ dissociation constant; MIANS, 2-(4 -maleimidylanilino)naphthalene-6-sulfonic acid; MOPS, 3-(N -morpholino) propanesulfonic acid; Quin-2, 2-[(2-amino-5-methylphenoxy)methyl]-6-methoxy-8-aminoquinoline-n,n,n ,n -tetraacetic acid; TFP, trifluoperazine dihydrochloride; Tn, troponin; TnC, troponin C; TnCdanz, TnC labeled on Met with 5-dimethylaminonaphthalene-1-sulfonyl aziridine; TnCEndog, endogenous TnC; TnCF29W, TnC containing the F29W mutation; TnCF29W(5OH), TnCF29W containing the Trp analog 5-fluoro-dl-tryptophan; TnCIAEDANS, TnC labeled at Cys 101 with IAEDANS; TnCTIA5A4CN,SC101S, TnC containing the T54C and C101S mutations labeled on T54C with IAANS; TnCTM5I4ACN,CS101S, TnC containing the T54C and C101S mutations labeled on T54C with MIANS; TnI, troponin I; TnI96–148, chicken skeletal troponin I peptide corresponding to residues 96–148; TnIAANS, Tn complex containing TnI labeled with IAANS. Fast-twitch skeletal muscle troponin C (TnC) contains two Nterminal and two C-terminal EF-hand Ca2+ binding sites that can be distinguished according to their Ca2+ dissociation rates. Based on data obtained from TnC in solution, only the N-terminal EF-hands release Ca2+ fast enough to be involved in the regulation of muscle relaxation (Johnson et al, 1981; Stephenson and Williams, 1981). This hypothesis has not been tested with TnC bound in the myofibril. Under normal physiological conditions it may be that no one mechanism is the all-inclusive rate-limiting step of relaxation, but that these processes may be working together (Johnson et al, 1997; Luo et al, 2002)
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