Abstract 2461 Illuminating Mg2+ in Living Cells Using a Novel Genetically Encoded Fluorescent Mg2+ Sensor

Mg2+ is the second most abundant cellular metal ion and can be found complexed by phosphonucleotides (e.g. ATP), key enzymes involved in central metabolism, and nucleic acids. Mg2+ has been implicated in cell cycle regulation, energy metabolism, and as a novel second messenger involved in cell signaling. However, the cellular distribution and molecular mechanisms involved in regulating cellular Mg2+ have remained elusive due to the scarcity of fluorescent tools to study Mg2+ in living cells. To this end, we developed two fluorescence resonance energy transfer (FRET)-based Mg2+ sensors that contain distinct Mg2+-binding domains (MBD) flanked by the same two fluorescent proteins: ECFP and cpVenus173. The MBDs interrogated in this study were derived from the EF hand proteins cardiac troponin C (cTnC) and the neuronal Ca2+-binding protein S-CaBP-1. In addition, we evaluated Centrin-3 (Cen3) - the EF hand protein that was used in the original FRET-based Mg2+ sensor MagFRET. We performed a side-by-side comparison of the in vitro dynamic range of all three sensors in response to Ca2+ and Mg2+ and found that there was little to no response for all three platforms. Truncation of the Cen3-based sensor resulted in a high dynamic range (4.24 ± 0.362). To assess Mg2+-selectivity compared with Ca2+, we titrated the truncated Cen3-based sensor with Mg2+ and Ca2+ (ranging from 1 μM – 10 mM) and determined the apparent dissociation constant (Kd') for Ca2+ and Mg2+ binding. We determined that the Kd' for Ca2+ and Mg2+ binding was 94.4 μM ± 28.0 and 1.60 mM ± 0.3768, respectively, indicating that the sensor had a higher affinity for Ca2+. In an effort to shift the affinity of the sensor toward Mg2+ and away from Ca2+, we targeted a glutamate residue within one of the EF hands, given its documented importance in Ca2+ coordination. We mutated this residue to either aspartate (E12D) or glutamine (E12Q). The truncated Cen3-based sensor containing E12D showed a 39.5-fold reduction in Ca2+ binding with a modest 3.7-fold decrease in Mg2+ affinity. Strikingly, E12Q reduced Ca2+ affinity by 24.5-fold with no apparent change in Mg2+ binding. The Cen3-based Mg2+ sensor containing both the truncation and E12Q mutation outperforms MagFRET-1 when expressed in the cytosol of HeLa cells and MIN6 cells. Current studies are focused on interrogating mitochondrial and endoplasmic reticulum Mg2+ using targeted versions of our optimized Mg2+ sensor platform. This work was supported by a NMU Faculty Research Grant to E.P. and an NMU Undergraduate Research Fellowship Award to W.K.