Binding Properties of the Calcium-Activated F2 Isoform of Lethocerus Troponin C

Muscle contraction is initiated by impulses from the motor neurons to the muscle that cause depolarization of the innervated fibers (see refs (1−3) for reviews). In synchronous muscles, an action potential triggers release of Ca2+ from an internal store within the fiber, the sarcoplasmatic reticulum. The released Ca2+ diffuses into the myofibrils, binds to control sites on the contractile filaments, and initiates contraction. This contraction is terminated when the sarcoplasmatic reticulum resequesters the released Ca2+ and reduces its cytoplasmatic concentration to a level below the threshold required for contractile activity. The Ca2+ sensor that regulates this process resides in the tropomyosin−troponin complex. In the absence of Ca2+, tropomyosin, a rodlike protein that extends over seven actin monomers, blocks the myosin-binding sites present on the thin filament (3−5). Troponin is a complex of three regulatory proteins: a Ca2+ binding protein (TnC), an inhibitory protein (TnI), and a tropomyosin binding protein (TnT) (6). TnC senses Ca2+ through up to four Ca2+ binding motifs (EF-hands) distributed across two globular domains, the N- and C-terminal domains (7−11). The C-terminal domain (C lobe) usually interacts with an N-terminal region of TnI and anchors it into a specific conformation (12−15). When the N-terminal domain (N lobe) of TnC is able to bind Ca2+, it undergoes a conformational transition that allows it to bind to the “switch peptide” of TnI (residues 115−131 of skeletal TnI) (12), initiating the tropomyosin movement that causes the generation of force. In some muscles, however, such as in the flight muscles of many insects, contraction is not simply regulated by the Ca2+ switch. The wing beat frequency is too high for individual contractions of the flight muscles to be activated by Ca2+16−19. To adapt to this requirement, indirect flight muscles (IFM) have evolved two distinct forms of activation, which are finely tuned to the type of movement required (20,21): a stretch-activated mechanism (asynchronous contraction) is used for flying, whereas Ca2+ regulation remains important during the “warm-up” contractions that precede flight in large insects (synchronous contraction). During flight, stretch and Ca2+ activation are finely balanced, depending on the Ca2+ concentration produced by the intermittent nerve impulses (20−22). Despite its importance for understanding the mechanisms of muscle contraction, relatively little is known about stretch activation and the way it coexists with synchronous regulation, although current evidence all seems to indicate that the muscle regulation in insects differs markedly from that of vertebrates. Most of our knowledge about insect muscles comes from studies of Lethocerus, a giant water bug of the Belostomatidae family, native to Southeast Asia, which is commonly used as a model system because its large muscle fibers are easily manageable. In Lethocerus, two isoforms of TnC coexist in the same myofibril, F1 and F2, in a ratio of ∼10:1 (23,24). F1 TnC, which has a single Ca2+ binding site in the C lobe, is responsible for activating the muscle following a stretch. F2 TnC has two Ca2+ binding sites, one in each lobe, and produces a sustained contraction, the magnitude of which is dependent on the Ca2+ concentration (20). In previous studies, we have shown that the C lobe of F1 TnC binds to peptides spanning the N-terminal and inhibitory regions of TnH [the insect equivalent of TnI (25)], although with very different affinities (26). The equilibrium dissociation constant for the complex with the N-terminal TnH peptide is in the nanomolar range, which is comparable to the values reported for the skeletal TnC−TnI complex (27,28). The affinity for the peptide from the inhibitory region is much lower, in the micromolar range, which is comparable to the affinity of the inhibitory peptide of skeletal TnI for the C lobe of TnC (29). Both interactions are essentially Ca2+ independent, and the N lobe of F1 TnC appears to play no role. It was therefore suggested that competition between two distinct regions of TnH, which alternately occupy the C lobe of F1 TnC, could be the basis of stretch activation. The relative affinity of F1 TnC and F2 TnC for the whole TnI sequence in TnH, in the presence of Ca2+, has been determined (24). The overall affinity of F2 TnC is greater than that of F1 TnC, which is likely caused by the two binding sites for TnI in F2 TnC and the single site in F1 TnC. Here, we have used a range of biophysical techniques to dissect the interaction of F2 TnC into specific contributions using synthetic peptides from TnH and studied how the complex affinities are modulated by the presence of Ca2+ and/or Mg2+. We show that F2 TnC binds to the same regions of TnH as F1 TnC but with two important differences. First, the interaction of F2 TnC with the peptides shows significant Ca2+ dependence, and second, interaction with the peptides does not involve just the TnC C lobe but causes a more generalized conformational change. Our data improve our understanding of Ca2+ regulation of insect flight muscle.