Intracellular coupling via limiting calmodulin.
Measurements of cellular Ca2+-calmodulin concentrations have suggested that competition for limiting calmodulin may couple calmodulin-dependent activities. Here we have directly tested this hypothesis. We have found that in endothelial cells the amount of calmodulin bound to nitric-oxide synthase and the catalytic activity of the enzyme both are increased approximately 3-fold upon changes in the phosphorylation status of the enzyme. Quantitative immunoblotting indicates that the synthase can bind up to 25% of the total cellular calmodulin. Consistent with this, simultaneous determinations of the free Ca2+ and Ca2+-calmodulin concentrations in these cells performed using indo-1 and a fluorescent calmodulin biosensor (Kd = 2 nm) indicate that increased binding of calmodulin to the synthase is associated with substantial reductions in the Ca2+-calmodulin concentrations produced and an increase in the [Ca2+]50 for formation of the calmodulin-biosensor complex. The physiological significance of these effects is confirmed by a corresponding 40% reduction in calmodulin-dependent plasma membrane Ca2+ pump activity. An identical reduction in pump activity is produced by expression of a high affinity (Kd = 0.3 nm) calmodulin biosensor, and treatment to increase calmodulin binding to the synthase then has no further effect. This suggests that the observed reduction in pump activity is due specifically to reduced calmodulin availability. Increases in synthase activity thus appear to be coupled to decreases in the activities of other calmodulin targets through reductions in the size of a limiting pool of available calmodulin. This exemplifies what is likely to be a ubiquitous mechanism for coupling among diverse calmodulin-dependent activities.
Division of Molecular Biology & Biochemistry, School of Biological Sciences, University of Missouri, Kansas City 64110-2499, USA.
Quang-Kim
QK
Tran
D J
DJ
Black
Anthony
A
Persechini
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism; D017136:Ion Transport; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D011485:Protein Binding
D002147:Calmodulin; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium