title: Characterization of troponin-C interactions in skinned barnacle muscle: comparison with troponin-C from rabbit striated muscle.
abstract: Previously it was shown that when troponin-C (TnC) is extracted from barnacle myofibrillar bundles they lose their Ca2+ sensitivity, which can be restored by adding back barnacle TnC (either isoform, BTnC1 or BTnC2). Thus barnacle muscle shows thin filament regulation, as does rabbit psoas skeletal muscle. In this paper we compare the interactions of barnacle and rabbit fast muscle TnC in their respective muscles. We demonstrate that muscle fibres from the giant barnacle, Balanus nubilus, contain about 186 microM kg-1 muscle tissue of BTnC1 plus BTnC2 compared to about 91 microM kg-1 of TnC in rabbit psoas muscle fibres. Extraction of BTnC is achieved using similar low ionic strength, low divalent ion Ca(2+)-low Mg2+ conditions which are required for TnC extraction in rabbit psoas skinned muscle fibres; extraction was prevented by 1 mM Mg2+. Full reconstitution of Ca(2+)-sensitivity was achieved by adding back BTnC (1 + 2, or 2). Reconstitution of barnacle muscle with rabbit fast skeletal TnC (RTnC) was more complex, with partial recovery of Ca(2+)-sensitivity with reconstitution in the presence of 3 mM Mg2+ and more fully with reconstitution in the presence of activating Ca2+ (pCa 4.0). This suggests that the barnacle TnC-TnI (troponin I) recognition sites may be more complex than in rabbit because the barnacle sites appear to have at least two different conformations or types, in which one recognizes RTnC in the presence of Mg2+ and the other only in the presence of Ca2+ and Mg2+. This is consistent with the presence of several TnI isoforms in barnacle striated myofibrils. RTnC has two C-terminal Ca(2+)-Mg2+ binding sites that are thought to be involved in the Mg(2+)-sensitive binding of RTnC in rabbit muscle, yet it has been suggested that this site in barnacle muscle does not bind Mg2+, even though Mg2+ stabilizes BTnC binding in barnacle muscle. Consistent with this stabilizing action of Mg2+, using fluorescent probes IAANS or IAE on isolated BTnC2 we demonstrate that BTnC2 binds both Ca2+ and Mg2+, but the data do not suggest direct competition. Consistent with the C-terminal sites on BTnC being Ca(2+)-specific, BTnC1 + 2 could only reconstitute low levels of force (about 1/3) in TnC-extracted rabbit skinned muscle fibers in the presence of pCa 4.0 (not just Mg2+) and only at low ionic strengths (0.09 M). Ca(2+)-activation of contraction was further examined using fluorescently labelled BTnC2 (labelled with IANBD) incorporated into skinned barnacle myofibrillar bundles. Maximal Ca2+ binding produced structural changes in BTnC which resulted in a 45% decrease in the fluorescence compared to the value at pCa 9.2. The magnitude of the fluorescence decrease paralleled the increase in force with increasing Ca2+. The Hill fits to the data gave pCa1/2 and n of 5.61 +/- 0.02 and 2.06 +/- 0.12 for force, and 5.52 +/- 0.02 and 1.88 +/- 0.10 for fluorescence. Removing MgATP to induce rigor in the fibre decreased BTnC2-NBD fluorescence only about 11%, but the addition of Ca2+ in rigor further decreased the fluorescence to a slightly larger extent than under maximal Ca2+ activating conditions. These fluorescence changes are qualitatively similar to the fluorescence enhancement seen with Ca(2+)-activation and rigor with RTnCDanz exchanged into rabbit psoas skinned muscle fibres. The data support a similar model for Ca(2+)-activation of force in barnacle muscle and in rabbit psoas skeletal muscle fibres.
authors.affiliation: Department of Physiology and Biophysics, University of Washington, Seattle 98195-7290, USA.
authors.forename: A M
authors.initials: AM
authors.lastname: Gordon
authors.forename: Y
authors.initials: Y
authors.lastname: Qian
authors.forename: Z
authors.initials: Z
authors.lastname: Luo
authors.forename: C K
authors.initials: CK
authors.lastname: Wang
authors.forename: R L
authors.initials: RL
authors.lastname: Mondares
authors.forename: D A
authors.initials: DA
authors.lastname: Martyn
mesh_terms: D000818:Animals; D005453:Fluorescence; D005456:Fluorescent Dyes; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D009210:Myofibrils / Q000737:chemistry; D011817:Rabbits; D001468:Thoracica / Q000737:chemistry / Q000378:metabolism*; D019209:Troponin C / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D019209:Troponin C