MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor

Abstract

Magnesium has important structural, catalytic and signaling roles in cells, yet few tools exist to image this metal ion in real time and at subcellular resolution. Here we report the first genetically encoded sensor for Mg2+, MagFRET-1. This sensor is based on the high-affinity Mg2+ binding domain of human centrin 3 (HsCen3), which undergoes a transition from a molten-globular apo form to a compactly-folded Mg2+-bound state. Fusion of Cerulean and Citrine fluorescent domains to the ends of HsCen3, yielded MagFRET-1, which combines a physiologically relevant Mg2+ affinity (K d = 148 µM) with a 50% increase in emission ratio upon Mg2+ binding due to a change in FRET efficiency between Cerulean and Citrine. Mutations in the metal binding sites yielded MagFRET variants whose Mg2+ affinities were attenuated 2- to 100-fold relative to MagFRET-1, thus covering a broad range of Mg2+ concentrations. In situ experiments in HEK293 cells showed that MagFRET-1 can be targeted to the cytosol and the nucleus. Clear responses to changes in extracellular Mg2+ concentration were observed for MagFRET-1-expressing HEK293 cells when they were permeabilized with digitonin, whereas similar changes were not observed for intact cells. Although MagFRET-1 is also sensitive to Ca2+, this affinity is sufficiently attenuated (K d of 10 µM) to make the sensor insensitive to known Ca2+ stimuli in HEK293 cells. While the potential and limitations of the MagFRET sensors for intracellular Mg2+ imaging need to be further established, we expect that these genetically encoded and ratiometric fluorescent Mg2+ sensors could prove very useful in understanding intracellular Mg2+ homeostasis and signaling.

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“…These sensors are based on proteins that can sense and bind the ion of interest with a high affinity. Upon binding, a conformational change occurs within the sensor, inducing or diminishing fluorescence or FRET [ 14 , 27 – 29 , 35 , 36 , 40 , 43 , 44 , 49 , 50 , 60 , 72 ]. b Overview of optogenetic signal transduction sensors.…”

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“…The strength of the specificity related to other molecules depends upon the sensory domain of the nanosensor that undergoes conformational changes by sensing and binding with the specific metabolites that cause a change in the FRET ratio. A similar approach has also been employed to measure the FRET ratio change of donor to acceptor emission intensity for monitoring the other nutrients and ions like phosphate [27], magnesium [28], and molybdenum [29]. Prior to estimating the equilibrium dissociation constant (K d ) for various periplasmic binding proteins, equilibrium dialysis was used, but in our study, we determined an affinity based on the FRET ratio change and increase in fluorescence induced by conformational changes in the sensor protein.…”

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“…Recently, Zhong and co‐workers enhanced chloride sensitivity, adjusted photostability, and reduced pH interference in a new GE‐YFP sensor . By taking advantage of FRET technology, the first GE fluorescent sensor for Mg 2+ was developed and named MagFRET . Alternatively, MagIC, another GE fluorescent indicator for monitoring Mg 2+ , was designed by using a non‐FRET imaging approach .…”

“…Along with consumption, cellular micronutrient uptake is a crucial factor determining micronutrient deficiency, which is still poorly understood. For instance, magnesium, which has important structural, catalytic, and signaling roles in cells, can be monitored in real time by using the FRET‐based genetically encoded sensor MagFRET‐1 . Combined with an integrated fluorescence sensor, this could prove very useful in understanding intracellular Mg 2+ homeostasis and signaling.…”