Measurements of the free luminal ER Ca2+ concentration with targeted “cameleon” fluorescent proteins

Abstract

The free ER Ca2+ concentration, [Ca2+]ER, is a key parameter that determines both the spatio-temporal pattern of Ca2+ signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca2+ activity within the ER is thus critical for our understanding of cell signaling. Such measurements, however, are particularly challenging given the highly dynamic nature of Ca2+ signals, the complex architecture of the ER, and the difficulty of addressing probes specifically into the ER lumen. Prompted by these challenges, a number of ingenious approaches have been developed over the last years to measure ER Ca2+ by optical means. The two main strategies used to date are Ca2+-sensitive synthetic dyes trapped into organelles and genetically encoded probes, based either on the photoprotein aequorin or on the green fluorescent protein (GFP). The GFP-based Ca2+ indicators comprise the camgaroo and pericam probes based on a circularly permutated GFP, and the cameleon probes, which rely on the fluorescence resonance energy transfer (FRET) between two GFP mutants of different colors. Each approach offers unique advantages and suffers from specific drawbacks. In this review, we will discuss the advantages and pitfalls of using the genetically encoded “cameleon” Ca2+ indicators for ER Ca2+ measurements.