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.
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, MO 64110-2499, USA.
D J
DJ
Black
Quang-Kim
QK
Tran
Anthony
A
Persechini
D015374:Biosensing Techniques / Q000379:methods; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002147:Calmodulin / Q000032:analysis* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007211:Indoles; D007700:Kinetics; D008164:Luminescent Proteins; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins; D012680:Sensitivity and Specificity
D002121:Calcium Channel Blockers; D002147:Calmodulin; D007211:Indoles; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C048960:indo-1; D002118:Calcium