CALPONIN-CALMODULIN INTERACTION - PROPERTIES AND EFFECTS ON SMOOTH AND SKELETAL-MUSCLE ACTIN-BINDING AND ACTOMYOSIN ATPASES

Smooth muscle calponin bound to the biologically active fluorescent ca lmodulin [2-(4'-maleimidoanilino)naphthalene-6-sulfonic acid.calmoduli n] (MIANS.CaM) with a K(d) of 80 nM and produced a 3.4-fold fluorescen ce enhancement. PKC-phosphorylated calponin (1.3 mol of P(i)/mol) boun d to CaM with approximately 15-fold lower affinity. Calponin inhibited CaM (10 nM) activation of the Ca2+-/CaM-activated cyclic nucleotide p hosphodiesterase (PDE) with an IC50 of 138 nM. The calponin-CaM intera ction was Ca2+-dependent: half-maximal binding of calponin to MIANS.Ca M occurred at pCa 6.6 with a Hill coefficient of 2.4. Stopped-flow flu orescence kinetic analysis demonstrated that EGTA chelation of Ca2+ fr om CaM disrupted the MIANS.CaM-calponin complex at a rate of 1 s-1. Ca lponin bound MIANS.CaM at a rate of (6.0 +/- 1.8) X 10(6) M-1 s-1, and melittin and unlabeled brain CaM both disrupted the MIANS.CaM-calponi n complex at a rate of 0.3 +/- 0.1 s-1. These studies suggest that cal ponin binds CaM with 80-fold lower affinity than myosin light-chain ki nase and that calponin associates with CaM much slower than it associa tes with caldesmon or myosin light-chain kinase. The physiological rel evance of the CaM-calponin interaction was evaluated by analysis of th e effects of Ca2+-CaM on (i) the interaction of calponin with actin an d (ii) calponin-mediated inhibition of actin-activated myosin MgATPase activity. Ca2+-CaM half-maximally inhibited calponin (2 muM) binding to smooth and skeletal muscle actins (9 muM) at 5.4 and 11 muM CaM, re spectively. Ca2+-CaM failed to reverse calponin inhibition of smooth o r skeletal muscle actin-activated myosin MgATPases, even at molar rati os of CaM that were supraphysiological relative to actin and calponin. Consistent with these findings, Ca2+-CaM, under ATPase reaction condi tions, failed to dissociate calponin from actin. We conclude that calp onin's physiological function (inhibition of myosin cross-bridge cycli ng) is probably not modulated by its interaction with CaM.