Effects of myosin light chain kinase and peptides on Ca2+ exchange with the N- and C-terminal Ca2+ binding sites of calmodulin

Abstract

Myosin light chain kinase and peptides from the calmodulin (CaM) binding domains of myosin light chain kinase (RS-20, M-13), CaM kinase II, and the myristoylated alanine-rich protein kinase C substrate protein slowed Ca2+ dissociation from CaM's N-terminal sites from 405 ± 75/s to 1.8-2.9/s and from CaM's C-terminal sites from 2.4 ± 0.2/s to 0.1-0.4/s at 10 °C. Since Ca2+ dissociates 5-29 times faster from the N-terminal in these CaM·peptide complexes and both lobes are required for activation, Ca2+ dissociation from the N-terminal would control target protein inactivation. Ca2+ binds 70 times faster to the N-terminal (1.6 × 108 M-1 s-1) than the C-terminal sites (2.3 × 106 M-1 s-1). In a 0.6-ms half-width Ca2+ transient, Ca2+ occupied >70% of the N-terminal but only 20% of the C-terminal sites. RS-20 produced a 9-fold and CaM kinase II a 6.3-fold increase in C-terminal Ca2+ affinity, suggesting that some target proteins may be bound to the C-terminal at resting [Ca2+]. When this is the case, Ca2+ exchange with the faster N-terminal sites may regulate CaM's activation and inactivation of these target proteins during a Ca2+ transient.