Figure 1.
Organic synthesis of KMG-301.
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.
Figure 3.
KMG-301 detects the changes in Mg2+ concentration in isolated mitochondria.
(A) Time-course and pseudo-colored images of the change in the fluorescence of KMG-301 in isolated mitochondria stained with KMG-301AM. Stepwise increases in the extramitochondrial Mg2+ concentration induced increases in the fluorescence of KMG-301 in mitochondria. This indicates that Mg2+ absorption in mitochondria was measured successfully with KMG-301. The scale bar indicates 10 µm. (B) Time-course and pseudo-colored images of the change in the fluorescence of KMG-301 in response to 5 µM FCCP. The FCCP–induced release of Mg2+ from mitochondria was observed successfully. (C) Mean fluorescence intensities of TMRE before stimuli, 5 min after FCCP treatment and 10 min after washout in isolated mitochondria from PC12 cells. The membrane potential partially recovered after washout of FCCP. (D) Mean fluorescence intensities of KMG-301 before stimuli, 5 min after FCCP treatment and 10 min after washout. Fluorescence of KMG-301 was increased upon repolarization of the mitochondrial membrane potential. This data indicates that the fluorescence of KMG-301 is responding to the changes in [Mg2+]mito. The error bars indicate SEM.
Figure 4.
KMG-301 is functional in mitochondria in living cells.
(A) Time-course of the changes in the fluorescence of KMG-301 in PC12 cells expressing negative control miR RNAi (gray line) and miR RNAi for Mrs2 (black line). An increase in the extracellular Mg2+ concentration from 0 mM to 10 mM induced an increase in the fluorescence of KMG-301 in PC12 cells expressing negative control miR RNAi. This increase in fluorescence was significantly suppressed in PC12 cells expressing miR RNAi for Mrs2. (B) Comparison of the relative fluorescence intensities of KMG-301 1 minute after the increase in extracellular Mg2+ concentration. Both two miR RNAis significantly inhibited the increase in fluorescence of KMG-301. This data indicates that KMG-301 was internalized in mitochondria and responded to changes in [Mg2+]mito. The error bars indicate SEM. * indicates p<0.05, and ** indicates p<0.01.
Figure 5.
KMG-301 was not released from mitochondria upon depolarization of their membrane potential.
(A) FCCP induced a decrease in fluorescence of rhodamine 123 in the cell body (presence of mitochondria, gray line). On the other hand, in the nucleus area (no mitochondria area), an increase in fluorescence intensity was observed (black line). It is caused by the release of the probe from mitochondria to the cytosol. These lines show the time-course average of 60 cells. (B) Whereas FCCP also induced a decrease in fluorescence of KMG-301 in the cell body (gray line), no increase in fluorescence was observed in the nucleus area (no mitochondria area, black line). This data indicates that KMG-301 is not released from mitochondria depending on the depolarization of their membrane potential. These lines show the time-course average of 43 cells. (C) Fluorescence images and pseudo-colored images (F/F0) of non-differentiated PC12 cells stained with Rh123 and KMG-301. Upon FCCP administration, increases in fluorescence in the nucleus area were observed only in Rh123 loaded cells. Red circles indicate the nucleus areas. The scale bar indicates 10 µm.
Figure 6.
Simultaneous measurements of cytosolic and mitochondrial Mg2+ concentration changes.
(A) Time-course of the changes in [Mg2+]cyto (gray line) and [Mg2+]mito (back line) was measured with KMG-104AM and KMG-301AM, respectively, in differentiated PC12 cells. Administration of 5 µM FCCP induced a rapid increase in [Mg2+]cyto and a gradual decrease in [Mg2+]mito. (B) Pseudo–colored images of changes in [Mg2+]cyto (upper) and [Mg2+]mito (bottom) were obtained at the indicated times. (C) Time-course of the changes in [Mg2+]cyto (gray line) and [Mg2+]mito (back line) in hippocampal neurons. Administration of 5 µM FCCP induced a gradual increase in [Mg2+]cyto and a rapid decrease in [Mg2+]mito. (D) Pseudo–colored images of changes in [Mg2+]cyto (upper) and [Mg2+]mito (bottom) were obtained at the indicated times. The error bars indicate SEM. The scale bars indicate 10 µm.
Figure 7.
Putative PD inducible substance caused Mg2+ release from mitochondria.
(A) Time-course of the change in [Mg2+]mito was measured with KMG-301 in differentiated PC12 cells. Administration of 5 mM MPP+ induced a gradual decrease of [Mg2+]mito. The error bars indicate SEM. (B) Comparison of the relative fluorescence intensities of KMG-301 10 minutes after the administration of MPP+ at the indicated concentrations. The [Mg2+]mito decreased in a dose-dependent manner. The error bars indicate SEM.