Monitoring the total available calmodulin concentration in intact cells over the physiological range in free Ca 2

• Published in: Cell calcium (Edinburgh) Vol. 35; no. 5; pp. 415 - 425
• Main Authors: Black, D.J, Tran, Quang-Kim, Persechini, Anthony
• Format: Journal Article
• Language: English
• Published: Elsevier India Pvt Ltd 2004
• Subjects: Biosensor; Ca 2+ binding; Calmodulin; Biosensor; Ca 2+ binding; Calmodulin
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/j.ceca.2003.10.005

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 K d value of 3 μM, 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 Ca 2+ concentrations to be monitored concurrently. We have determined an intracellular available calmodulin concentration of 8.8±2.2 μM under resting conditions in a human kidney cell line stably expressing the biosensor. Elevation of the intracellular free Ca 2+ 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 Ca 2+ concentration fits a consumption curve with a cooperativity coefficient of 1.8 and a [Ca 2+] 50 of 850 nM. There is no detectible binding of calmodulin to the biosensor above a free Ca 2+ concentration of ∼4 μM, consistent with an available calmodulin concentration ≤200 nM under these conditions, and an overall excess of calmodulin-binding sites.