Novel fluorescent indicator proteins for monitoring free intracellular Ca2+.

We have recently described a fluorescent indicator protein in which red- and blue-shifted variants of green fluorescent protein are joined by the calmodulin-binding sequence from smooth muscle myosin light chain kinase [Romoser V.A., Hinkle P.M., Persechini A. Detection in living cells of Ca(2+)-dependent changes in the fluorescence of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. A new class of fluorescent indicators. J Biol Chem 1997; 272: 13270-13274]. The fluorescence emission of this protein at 505 nm (380 nm excitation) is reduced by approximately 65% when (Ca2+)4-calmodulin is bound, with a proportional increase in fluorescence emission at 440 nm. We have found that fusion of an engineered calmodulin, in which the C- and N-terminal EF hand pairs have been exchanged, to the C-terminus of this protein results in a novel indicator that responds directly to changes in the Ca2+ ion concentration, with an apparent Kd value of 100 nM for Ca2+ in the presence of 0.5 mM Mg2+. The affinity of the indicator for Ca2+ can be decreased by altering the amino acid sequence of the calmodulin-binding sequence to weaken its interaction with the intrinsic calmodulin domain. The fluorescence emission of this indicator can be used to monitor physiological changes in the free Ca2+ ion concentration in living cells.