We have previously described fluorescent indicators for Ca2+ (FIP-CAs) and (Ca2+)4-calmodulin (FIP-CBs) whose responses are based on a ligand-dependent decrease in fluorescence energy transfer (FRET) between GFP variants. The indicators for (Ca2+)4-calmodulin contain calmodulin-binding domains, those for Ca2+ also contain an integral calmodulin (CaM) domain. We have developed new versions of these indicators constructed with enhanced blue- and red-shifted GFPs suitable for stable and transient expression in mammalian cells, and have begun to use them to investigate the relationships between the free intracellular concentrations of Ca2+ ([Ca2+]i) and (Ca2+)4-CaM ([(Ca2+)4-CaM]i). When the blue-shifted fluorophore is excited at 380 nm these constructs exhibit an emission peak at 505 nm due to FRET to the red-shifted fluorophore.

We have made FIP-CBs with dissociation constants for (Ca2+)4-CaM of 0.5 nM, 20 nM, 300 nM and > 20 μM by introducing R →Q substitutions in the CaM-binding sequence, and have stablyexpressed them in HEK-293 cells (Fig. 1).