Cross-bridges affect both TnC structure and calcium affinity in muscle fibers.

In vertebrate striated muscle, calcium binding to troponin initiates contraction, a strong interaction of actin and myosin. In isolated proteins and skinned fibers, the strong interaction of myosin with actin also affects troponin. Fluorescent labels attached to troponin C show structural changes in the TnC environment with cross-bridge attachment and also with calcium binding. Evidence that this effect of crossbridges also occurs in intact striated muscle comes from studies in partially activated cardiac or skeletal muscle by others and in barnacle muscle by us. Length changes which detach myosin cross-bridges produce a brief burst of extra calcium that can be detected by aequorin in activated, voltage clamped single barnacle muscle fibers. That this calcium is coming from calcium bound to the activating site (troponin-C) is supported by several pieces of evidence. Studies on the dependence of the extra calcium on force and the time of the length change are consistent with the amplitude of the extra calcium being proportional to the bound calcium (CaTnC) and with increased cross-bridge attachment and force increasing calcium binding to troponin-C by up to a factor of 10. Importantly, stretch of active muscle (which first detaches cross-bridges and then enhances steady force) gives a biphasic response: first extra calcium (presumably due to cross-bridge detachment) and then, decreased calcium (presumably due to enhanced calcium binding to TnC). The enhanced calcium binding we see with elevated force (via strained cross-bridges) implies that calcium binding to TnC is enhanced not only be cross-bridge attachment but also by crossbridge (or thin filament) strain. This effect of cross-bridge attachment/force on calcium binding is consistent with a dual mechanism of calcium activation of contraction. First, calcium binds to troponin in the thin filament activating strong myosin binding to the thin filament. Then, strong myosin binding in turn provides additional activation either by increasing calcium binding or by changing the thin filament structure directly allowing additional cross-bridge attachment.