Dynamic and quantitative Ca2+ measurements using improved cameleons

Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation mic roscopy, they offer great promise for monitoring Ca2+ in whole organisms, t issues, organelles, and submicroscopic environments in which measurements w ere previously impossible. However, the original cameleons suffered from si gnificant pH interference, and their Ca2+-buffering and cross-reactivity wi th endogenous CaM signaling pathways was uncharacterized. We have now great ly reduced the pH-sensitivity of the cameleons by introducing mutations V68 L and Q69K into the acceptor yellow green fluorescent protein. The resultin g new cameleons permit Ca2+ measurements despite significant cytosolic acid ification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused tog ether in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpres sed, the primary effect is likely to be the unavoidable increase in Ca2+ bu ffering rather than specific perturbation of CaM-dependent signaling.