Ca2+-calmodulin binding to caldesmon and the caldesmon-actin-tropomyosin complex. Its role in Ca2+ regulation of the activity of synthetic smooth-muscle thin filaments

Abstract

We measured the concentration of calmodulin required to reverse inhibition by caldesmon of actin-activated myosin MgATPase activity, in a model smooth-muscle thin-filament system, reconstituted in vitro from purified vascular smooth-muscle actin, tropomyosin and caldesmon. At 37°C in buffer containing 120 mM-KCl, 4 μM-Ca2+-calmodulin produced a half-maximal reversal of caldesmon inhibition, but more than 300 μM-Ca2+-calmodulin was necessary at 25°C in buffer containing 60 mM-KCl. The binding affinity (K) of caldesmon for Ca2+-calmodulin was measured by a fluorescence-polarization method: K = 2.7 x 106 M-1 at 25°C (60 mM-KCl); K = 1.4 x 106 M-1 at 37°C in 70 mM-KCl-containing buffer; K = 0.35 x 106 M-1 at 37°C in 120 mM-KCl- containing buffer (pH 7.0). At 37°C/120 mM-KCl, but not at 25°C/60 mM-KCl, Ca2+-calmodulin bound to caldesmon bound to actin-tropomyosin (K = 2.9 x 106 M-1). Ca2+ regulation in this system does not depend on a simple competition between Ca2+-calmodulin and actin for binding to caldesmon. Under conditions, (37°C/120 mM-KCl) where physiologically realistic concentrations of calmodulin can Ca2+-regulate synthetic thin filaments, Ca2+-calmodulin reverses caldesmon inhibition of actomyosin ATPase by forming a non-inhibited complex of Ca2+-calmodulin-caldesmon-(actin-tropomyosin).