Newly Developed Mg2+–Selective Fluorescent Probe Enables Visualization of Mg2+ Dynamics in Mitochondria
Figure 2
(A) Absorbance and normalized fluorescence emission spectra of KMG-301 at different Mg2+ concentrations. Spectra were measured at a probe concentration of 5 µM at pH 7.2 (100 mM HEPES buffer). Mg2+ concentrations are 0 (black line) and 100 mM (gray line) for absorbance spectra, and from 0 mM to 100 mM for fluorescence spectra (excitation 540 nm). (B) Relative fluorescence intensities of KMG-301 in the presence of different cations in concentrations ranging from 0.1 to 200 mM (Mg2+, Ca2+), and from 0.1 to 500 mM (Na+, K+) normalized by the value in ion–free solution. (C) Metal ion selectivity of KMG-301 at physiological concentrations. Ni2+, Mn2+, Co2+, Cu2+, Zn2+, Fe2+ and Fe3+ were added at 1 µM. Ca2+ was added at 1 mM. Na+, K+ and Mg2+ were added at 100 mM. The concentrations were chosen according to the physiological conditions. Fluorescence intensities were normalized by the value in ion–free solution. (D) Effect of the pH on the fluorescence intensity of KMG-301 was measured at pH 5.5–6.5 (in 100 mM MES buffer) and pH 7.0–9.5 (in 100 mM HEPES buffer), with 0 mM or 100 mM of Mg2+. The fluorescence intensities were normalized by the value at pH 7.0 with 100 mM of Mg2+. (E) Localization of KMG-301 to mitochondria was confirmed in differentiated PC12 cells (upper) and rat hippocampal neurons (bottom). The localization of the fluorescence of KMG-301 (red) and MitoTracker Green FM (green) were corresponded to each other in both cells (merge). The scale bars indicate 10 µm.