The Mechanism of Smooth Muscle Caldesmon-Tropomyosin Inhibition of the Elementary Steps of the Actomyosin ATPase

Abstract

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-S1⅐ADP; however the rate of phosphate release from the actin-S1⅐ADP⅐P 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.Smooth muscle contractility and actomyosin ATP hydrolysis are regulated by Ca 2ϩ at two levels. The first involves phosphorylation of the myosin light chain by myosin light chain kinase upon stimulation by Ca 2ϩ -calmodulin. The second occurs via the thin filament-associated protein caldesmon. Caldesmon (CaD) 2 binds to actin, calmodulin, and tropomyosin (1) and inhibits the actin activation of both smooth and skeletal muscle myosin ATPase in vitro (2) and force production when added to smooth and skeletal muscle fibers (3-5). Caldesmon also regulates the actin activation of myosin I ATPase in smooth muscle (6). The smooth muscle thin filament is Ca 2ϩ -regulated and is made up of actin, tropomyosin, caldesmon, and a Ca 2ϩ -binding protein in molar ratios ϳ14:2:1:1 (7). Previous studies of the molecular mechanism of caldesmon inhibition of the actomyosin ATPase have focused on its effect on the ATPase steady state parameters K m and V max (8) and on the equilibrium binding of myosin to actin filaments (9 -12). A detailed mechanism by which caldesmon regulates the actin activation of myosin ATPase and force production has not been demonstrated by such experiments but two potentially conflicting models have been developed.A competition model proposes that ATPase inhibition by caldesmon is caused by a reduction in the formation of the weakly bound actomyosin complex as a...

Caldesmon and the Regulation of Cytoskeletal Functions

“…126 On the other hand, it was argued that the reduced binding rate could be balanced out by a change in the dissociation rate, yielding a moderate (9%) overall maximum rate reduction that fails to account for the observed level of inhibition (75%) of the ATPase unless a higher concentration of CaD was used. 128 The discrepancy is difficult to reconcile, not only because of the subtle differences in the experimental conditions (such as ionic strength and temperature), but also due to the intrinsically complicated nature of the system that involves multiple, mutually interacting, protein species. However, it might be worthwhile to point out that the cellular contents of CaD and actin only give an averaged ratio between the two.…”

“…This CooperativeAllosteric Model is supported by both structural 107 and biochemical data. 127,128 Two important pieces of evidence are that at low ratios (1:14) to actin, CaD was able to compete with not only weak, but also strong, myosin binding to actin 131 and that CaD was able to decrease the population of moving filaments without much changing the velocity in the in vitro motility assay. 132 These observations are indeed difficult to explain by the pure Competition Model.…”

“…While in many cases CaD behaves similarly to Tn, there are differences. Kinetic experiments (using skeletal S1) 128 showed that CaD affects the cross-bridge cycling by mainly slowing down the rate of phosphate release, rather than that of the ADP release as in the case of TnTm. 133 These results also suggested that CaD moves Tm between two states (open and closed), rather than three states (including a blocked state) as in the skeletal muscle system.…”

Role of Caldesmon in the Ca2+ Regulation of Smooth Muscle Thin Filaments

“…For experiments using low actin concentrations (below 4 M) actin was stabilized with phalloidin and used within 20 min of actin sample preparation (28). S1 was treated with N-ethyl maleimide as described by Swartz and Moss (33), and sheep aorta caldesmon was labeled with […”

“…These findings are compatible with the proposed mechanism. More recently we have investigated thoroughly the effect of caldesmon on the elementary steps of the actomyosin ATPase (28). We found that caldesmon had very little effect on the rate of S1 binding to actin-tropomyosin, acto-S1 dissociation by ATP and the rate of ADP release.…”