Measurements of the free luminal ER Ca 2+ concentration with targeted “cameleon” fluorescent proteins

Abstract

The free ER Ca 2+ concentration, [Ca 2+ ] ER , is a key parameter that determines both the spatio-temporal pattern of Ca 2+ signals as well as the activity of ER-resident enzymes. Obtaining accurate, time-resolved measurements of the Ca 2+ 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 Ca 2+ 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 Ca 2+ by optical means. The two main strategies used to date are Ca 2+ -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 Ca 2+ 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" Ca 2+ indicators for ER Ca 2+ measurements.

Ca2+ homeostasis in the endoplasmic reticulum measured with a new low-Ca2+-affinity targeted aequorin

“…Therefore, the data obtained with the old probe were necessarily an underestimation of the real value. Regarding the data obtained at 22 [19] to near 1 mM [20], with most of the obtained figures around 400-800 M [21][22][23][24][25][26][27]. On the other hand, NMR measurements [21], skeletal muscle cells [25,27] and rabbit heart cells [28] The new probe has also a very important technical and experimental advantage over the previous aequorin probes.…”

Ca2+ signaling, genes and the cell cycle

“…Intracellular Ca 2+ signaling is possible because cells maintain a low Ca 2+ background in the cytoplasm with concentrations of ∼100 nM. Ca 2+ signals are generated due to Ca 2+ influx from the extracellular space with concentration around 1-2 mM, or Ca 2+ release from intracellular Ca 2+ stores, primarily the endoplasmic reticulum (ER) with concentrations of 250-600 μM [6]. Channels and pumps on the ER and plasma membranes coordinately regulate Ca 2+ homeostasis in the ER and cytoplasm.…”

Some assembly required: Constructing the elementary units of store-operated Ca2+ entry

“…Knockdown of STIM1 suppresses SOCE, and EF-hand mutants of STIM1, designed to have low Ca 2+ affinity and thereby mimic the store-depleted state, activate I CRAC even when stores are full [39,40,44,45]. The isolated EF-SAM domain has a Ca 2+ binding affinity of 200-600 μM [46], overlapping the range of [Ca 2+ ] i reported for the ER lumen by fluorescent protein measurements [47].…”