Dominant affectors in the calmodulin network shape the time courses of target responses in the cell

In endothelial cells nitric oxide synthase is a dominant affector in the calmodulin network by virtue of its ability to bind a significant fraction of limiting intracellular calmodulin. We have investigated how this affector function influences the kinetics of calmodulin-dependent signaling in cells co-expressing the synthase and a fluorescent calmodulin target analog similar in its interactions with calmodulin to myosin light chain kinase. The synthase binds (Ca2+)4-calmodulin with a Kd value of ∼0.2 nM and an association rate constant of ∼1.5 × 105 M−1 s−1. These values are, respectively, 10- and 100-fold smaller than the corresponding values for the analog. Thus, when Ca2+ is added to a mixture of calmodulin, target analog and synthase in vitro a large fluorescence transient with a relaxation time of ∼600 s is observed as (Ca2+)4-calmodulin is rapidly bound to the analog and then slowly captured by the higher affinity synthase. A rapid increase in the free Ca2+ concentration elicits similar transient analog responses in cells expressing the cytoplasmic target analog and either a wild-type membrane bound or mutant cytoplasmic synthase. Transient responses are not observed in cells co-expressing the fluorescent analog and a mutant T497D synthase unable to bind calmodulin. These results demonstrate that dominant affectors in the calmodulin network shape both the magnitudes and time courses of target responses in the cell.