Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca2+

We describe the design, characterization and application of a new genetically encoded fluorescent biosensor for intracellular detection of both free Ca(2+)-calmodulin and apocalmodulin, which together comprise the available calmodulin concentration. The biosensor binds both forms of calmodulin with an apparent Kd value of 3 microM, and has kinetic properties making it suitable for monitoring dynamic changes on a subsecond time scale. It can be used in conjunction with the fluorescent Ca(2+)-indicator, indo-1, allowing the available calmodulin and free Ca2+ concentrations to be monitored concurrently. We have determined an intracellular available calmodulin concentration of 8.8 +/- 2.2 microM under resting conditions in a human kidney cell line stably expressing the biosensor. Elevation of the intracellular free Ca2+ concentration by agonist, store-operated Ca(2+)-entry or ionophore results in Ca(2+)-dependent consumption of the available calmodulin. A plot of normalized values for the available calmodulin concentration versus the free Ca2+ concentration fits a consumption curve with a cooperativity coefficient of 1.8 and a [Ca2+]50 of 850 nM. There is no detectible binding of calmodulin to the biosensor above a free Ca2+ concentration of approximately 4 microM, consistent with an available calmodulin concentration < or = 200 nM under these conditions, and an overall excess of calmodulin-binding sites.