NOVEL FLUORESCENT INDICATOR PROTEINS FOR MONITORING FREE INTRACELLULAR CA2+

We have recently described a fluorescent indicator protein in which re d-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 Ca2+-dependent changes in the fluorescence of an indicator co mposed of two green fluorescent protein variants linked by a calmoduli n-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 similar to 65% when (Ca2+)(4 )-calmodulin is bound, with a proportional increase in fluorescence em ission at 440 nm. We have found that fusion of an engineered calmoduli n, in which the C- and N-terminal EF hand pairs have been exchanged, t o the C-terminus of this protein results in a novel indicator that res ponds directly to changes in the Ca2+ ion concentration, with an appar ent K-d value of 100 nM for Ca2+ in the presence of 0.5 mM Mg2+. The a ffinity of the indicator for Ca2+ can be decreased by altering the ami no acid sequence of the calmodulin-binding sequence to weaken its inte raction with the intrinsic calmodulin domain. The fluorescence emissio n of this indicator can be used to monitor physiological changes in th e free Ca2+ ion concentration in living cells.