Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence - A new class of fluorescent indicators

We have designed a novel fluorescent indicator composed of two green fluorescent protein variants joined by the calmodulin-binding domain from smooth muscle myosin light chain kinase. When (Ca2+)(4)-calmodulin is bound to the indicator (K-d = 0.4 nM), fluorescence resonance energy transfer between the two fluorophores is attenuated; the ratio of the fluorescence intensity measured at 505 nm to the intensity measured at 440 nm decreases B-fold. Images of microinjected living cells demonstrate that emission ratios can be used to monitor spatio temporal changes in the fluorescence of the indicator, Changes in indicator fluorescence in these cells are coupled with no discernible lag(<1 s) to changes in the cytosolic free Ca2+ ion concentration, ranging from below 50 nM to similar to 1 mu M. This observation suggests that the activity of a calmodulin target with a typical 1 nM affinity for (Ca2+)(4)-calmodulin is responsive to changes in the intracellular Ca2+ concentration over the physiological range. It is likely that the indicator we describe can be modified to detect the levels of ligands and proteins in the cell other than calmodulin.