Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED)

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.853

Regulation of Contraction in Striated Muscle

Cardiac and skeletal muscle contraction are activated by Ca2+ binding to specific regulatory sites on the striated muscle thin filament. The thin filament is a large allosteric assembly, containing multiple copies of actin, tropomyosin, and the three troponin subunits (troponin C, troponin I, and troponin T). This review describes recent developments in understanding the structure and dynamics of these proteins and how they function to regulate contraction. Emphasis is placed on the cardiac thin filament and on the features that distinguish it from the skeletal muscle system. The discussion also emphasizes current knowledge of the protein-protein interactions involved in the cooperative processes of thin filament assembly and regulation.

Thin Filament-Mediated Regulation of Cardiac Contraction

(1984). Thin Filament Proteins and Thin Filament-Linked Regulation of Vertebrate Muscle Contractio. Critical Reviews in Biochemistry: Vol. 16, No. 3, pp. 235-305.

Thin filament proteins and thin filament-linked regulation of vertebrate muscle contraction.

Approximate Model of Cooperative Activation and Crossbridge Cycling in Cardiac Muscle Using Ordinary Differential Equations

Inhibition of actomyosin ATPase activity by troponin-tropomyosin without blocking the binding of myosin to actin.

The transient-state kinetics of binding of myosin subfragment 1 (SF-1) to regulated actin in the presence and absence of Ca2+ were investigated. The binding of SF-1 to pure actin, to actin-tropomyosin (actin-TM), or to actin-tropomyosin-troponin (actin-TM-TN) in the presence of Ca2+ was kinetically the same. In each case, the light-scattering transients were biphasic, suggesting a two-step binding of SF-1 to actin. Binding of SF-1 to regulated actin in the absence of Ca2+ was different from binding in its presence and also varied depending on whether SF-1 or regulated actin was in excess. The kinetic results in the absence of CA2+ are explained by a cooperative binding model, in which the initial binding of SF-1 molecules to open (active) actin sites increases the number of open sites. TN-I labeled with the fluorophore 4-(N-iodoacetoxyethyl-N-methyl)-7-nitrobenz-2-oxa-1,3 diazole (TN*) was used to probe the state of the actin-TM-TN complex. Binding of SF-1 or CA2+ to regulated actin (in the absence of Ca2+) decreased the fluorescence of actin-TM-TN* by 30%, suggesting that binding of SF-1 or CA2+ induces a similar change in state. The change in fluorescence of TN* was also used to measure the rate of the transition from the active to the relaxed state in the absence of CA2+, which was 430 sec-1 at 4 degrees C in 0.1 M KCl. The lag prior to association of SF-1 with regulated actin (in the absence of Ca2+) was abolished when three SF-1 molecules were prebound per seven G-actin monomers. Similarly, a titration of actin-TM-TN* (in the absence of Ca2+) with SF-1 or SF-1-ADP showed that most actin sites are open, as measured by the fluorescence change, when the occupancy of actin-TM-TN* by SF-1-ADP or SF-1 is approximately 50%. The evidence shows that partial occupancy of a block of G-actin sites (possibly seven) by SF-1 or SF-1-ADP stabilizes the open (active) conformation.

K M Trybus

Papers

Kinetic studies of the cooperative binding of subfragment 1 to regulated actin.