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Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca2+-binding protein and their interaction with myosin is Ca2+-regulated. We suggested that Ca2+ regulation by caldesmon and Ca2+-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle αα-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca2+-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca2+-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca2+-calmodulin-caldesmon activation of actin-tropomyosin.

Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca(2+)-binding protein and their interaction with myosin is Ca(2+)-regulated. We suggested that Ca(2+) regulation by caldesmon and Ca(2+)-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle alphaalpha-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca(2+)-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca(2+)-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca(2+)-calmodulin-caldesmon activation of actin-tropomyosin.

Caldesmon is a component of smooth muscle thin filaments that inhibits the actomyosin ATPase via its interaction with actin-tropomyosin. We have performed a comprehensive transient kinetic characterization of the actomyosin ATPase in the presence of smooth muscle caldesmon and tropomyosin. At physiological ratios of caldesmon to actin (1 caldesmon/7 actin monomers) actomyosin ATPase is inhibited by about 75%. Inhibitory caldesmon concentrations had little effect upon the rate of S1 binding to actin, actin-S1 dissociation by ATP, and dissociation of ADP from actin-S1 x ADP; however the rate of phosphate release from the actin-S1 x ADP x P(i) complex was decreased by more than 80%. In addition the transient of phosphate release displayed a lag of up to 200 ms. The presence of a lag phase indicates that a step on the pathway prior to phosphate release has become rate-limiting. Premixing the actin-tropomyosin filaments with myosin heads resulted in the disappearance of the lag phase. We conclude that caldesmon inhibition of the rate of phosphate release is caused by the thin filament being switched by caldesmon to an inactive state. The active and inactive states correspond to the open and closed states observed in skeletal muscle thin filaments with no evidence for the existence of a third, blocked state. Taken together these data suggest that at physiological concentrations, caldesmon controls the isomerization of the weak binding complex to the strong binding complex, and this causes the inhibition of the rate of phosphate release. This inhibition is sufficient to account for the inhibition of the steady state actomyosin ATPase by caldesmon and tropomyosin.