MagFRET: The First Genetically Encoded Fluorescent Mg2+ Sensor

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 (Kd = 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 (Kd 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.镁在细胞中发挥着重要的结构、催化与信号传导功能，但目前能够以实时、亚细胞分辨率对该金属离子进行成像的工具仍十分匮乏。本文报道了首个基因编码的Mg²+传感器MagFRET-1。该传感器基于人中心蛋白3（HsCen3）的高亲和力Mg²+结合结构域，该结构域可从熔球态脱辅基形式转变为紧密折叠的Mg²+结合状态。将Cerulean与Citrine荧光结构域融合至HsCen3的两端，即可得到MagFRET-1，该传感器兼具生理相关的Mg²+亲和力（解离常数Kd=148 μM），且因Cerulean与Citrine之间的荧光共振能量转移（FRET）效率发生改变，在结合Mg²+后发射比值提升50%。对金属结合位点进行突变后，得到了一系列MagFRET变体，其Mg²+亲和力相较于MagFRET-1减弱2至100倍，因此可覆盖宽泛的Mg²+浓度区间。在HEK293细胞中开展的原位实验表明，MagFRET-1可被靶向定位于细胞质与细胞核。当用洋地黄皂苷透化表达MagFRET-1的HEK293细胞时，可观测到其对胞外Mg²+浓度变化的清晰响应；而完整细胞则未出现类似变化。尽管MagFRET-1对Ca²+同样具有敏感性，但其亲和力已被充分减弱（Kd=10 μM），足以使该传感器不受HEK293细胞中已知Ca²+刺激的影响。虽然这类MagFRET传感器用于细胞内Mg²+成像的潜力与局限仍有待进一步探明，但我们预计这些基因编码的比率型荧光Mg²+传感器将为理解细胞内Mg²+稳态与信号传导提供极具价值的研究工具。