Dynamic and quantitative Ca 2+ measurements using improved cameleons

• DOI: 10.1073/pnas.96.5.2135
• Publication Date: 2002-07-26T14:39:15Z
• Authors: Atsushi Miyawaki; Oliver Griesbeck; Roger Heim; Roger Y. Tsien
• Keywords: Organelle; Calcium Signaling

Cameleons are genetically-encoded fluorescent indicators for Ca 2+ based on green protein variants and calmodulin (CaM). Because cameleons can be targeted genetically imaged by one- or two-photon excitation microscopy, they offer great promise monitoring in whole organisms, tissues, organelles, submicroscopic environments which measurements were previously impossible. However, the original suffered from significant pH interference, their -buffering cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced pH-sensitivity of introducing mutations V68L Q69K into acceptor yellow protein. The resulting new permit despite cytosolic acidification. When is elevated, CaM-binding peptide fused together a cameleon predominantly interact each other rather than free CaM-dependent enzymes. Therefore, if overexpressed, primary effect likely to unavoidable increase buffering specific perturbation signaling.