Dynamic and quantitative Ca ²⁺ measurements using improved cameleons

TLDR

Cameleons are genetically encoded fluorescent Ca²⁺ indicators based on GFP variants and calmodulin, enabling one‑ or two‑photon imaging of calcium in diverse biological contexts, but earlier versions suffered from pH sensitivity and uncharacterized Ca²⁺ buffering and cross‑reactivity with endogenous CaM signaling. In elevated Ca²⁺, the cameleon’s fused calmodulin and binding peptide preferentially interact with each other, reducing competition with free CaM and CaM‑dependent enzymes. Mutations V68L and Q69K in the acceptor YFP greatly reduce pH sensitivity, allowing accurate Ca²⁺ measurements even during cytosolic acidification, and overexpression mainly increases Ca²⁺ buffering without significantly perturbing CaM‑dependent signaling.

Abstract

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