The relationship between the free concentrations of Ca2+ and Ca2+- calmodulin in intact cells

Abstract

Using stably expressed fluorescent indicator proteins, we have determined for the first time the relationship between the free Ca2+ and Ca2+-calmodulin concentrations in intact cells. A similar relationship is obtained when the free Ca2+ concentration is externally buffered or when it is transiently increased in response to a Ca2+-mobilizing agonist. Below a free Ca2+ concentration of 0.2 μM, no Ca2+-calmodulin is detectable. A global maximum free Ca2+-calmodulin concentration of ~45 nM is produced when the free Ca2+ concentration exceeds 3 μM, and a half-maximal concentration is produced at a free Ca2+ concentration of 1μM. Data for fractional saturation of the indicators suggest that the total concentration of calmodulin-binding proteins is ~2-fold higher than the total calmodulin concentration. We conclude that high-affinity calmodulin targets (K(d) ≤ 10 nM) are efficiently activated throughout the cell, but efficient activation of low-affinity targets (K(d) ≥ 100 nM) occurs only where free Ca2+- calmodulin concentrations can be locally enhanced.