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Measurement of matrix free Mg2+ concentration in rat heart mitochondria by using entrapped fluorescent probes
G A Rutter1, N J Osbaldeston, J G McCormack
1Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Abstract:
1. The concentration of free Mg2+ ([Mg2+]m) within the matrix of isolated rat heart mitochondria was measured after loading of the mitochondria with the fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2. No detectable change in total mitochondrial magnesium content occurred during loading with the indicators. Apparent Kd values for Mg2+ of 3.7 mM and 2.3 mM were obtained for mag-indo-1 and mag-fura-2 respectively within mitochondria permeabilized to bivalent cations with ionomycin and the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These values are 2.7- and 1.8-fold greater respectively than those obtained for the free acid forms of the dyes in incubation medium. 2. Based on the above Kd values, mitochondrial matrix Mg2+ concentrations were found to lie in the range 0.8-1.5 mM in the absence, or immediately after the addition, of a respiratory substrate. 3. Incubation of mitochondria in the presence of respiratory substrate, but in the absence of external Mg2+, led to a time-dependent decline in [Mg2+]m to about half the initial values after 5 min. This was accompanied by a fall in the total mitochondrial magnesium content from 12.7 to 7.0 nmol/mg of protein. 4. ADP (0.5 mM), ATP (0.5 mM) or 10 mM-NaCl had no significant effect on the fall in [Mg2+], whereas 1 microM-nigericin blocked, and 0.3 microM-valinomycin accelerated, the fall. 5. External Mg2+ concentrations above 1 mM progressively inhibited and reversed the decline in free and total mitochondrial Mg2+.
Insights
Mitochondrial matrix free Mg2+ ([Mg2+]m) concentrations were measured using fluorescent indicators. Matrix Mg2+ levels were found to be 0.8-1.5 mM, declining when external Mg2+ was absent during respiration.
Area of Science:
- Mitochondrial Physiology
- Biochemistry
- Cellular Magnesium Homeostasis
Background:
- Mitochondrial magnesium ([Mg2+]) plays a crucial role in cellular energy metabolism and enzyme function.
- Accurate measurement of intramitochondrial free Mg2+ ([Mg2+]m) is essential for understanding its physiological roles.
- Fluorescent indicators like mag-indo-1 and mag-fura-2 are commonly used but require calibration within the mitochondrial matrix.
Purpose of the Study:
- To accurately quantify the concentration of free Mg2+ within the matrix of isolated rat heart mitochondria.
- To determine the apparent dissociation constants (Kd) of Mg2+ for mag-indo-1 and mag-fura-2 within the mitochondrial matrix.
- To investigate the dynamics of mitochondrial Mg2+ under varying respiratory and ionic conditions.
Main Methods:
- Isolated rat heart mitochondria were loaded with fluorescent Mg2+ indicators mag-indo-1 and mag-fura-2.
- Mitochondria were permeabilized using ionomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone to calibrate indicator Kd values.
- Matrix free Mg2+ ([Mg2+]m) was measured using spectrofluorometry under different substrate and ion conditions.
Main Results:
- Apparent Kd values for Mg2+ were determined to be 3.7 mM for mag-indo-1 and 2.3 mM for mag-fura-2 within the mitochondrial matrix.
- Baseline mitochondrial matrix Mg2+ concentrations ([Mg2+]m) were established at 0.8–1.5 mM in the absence of respiratory substrate.
- Incubation with respiratory substrate but without external Mg2+ caused a time-dependent decrease in [Mg2+]m and total mitochondrial magnesium.
- Nigericin blocked, while valinomycin accelerated, the decline in [Mg2+]m; external Mg2+ > 1 mM inhibited and reversed this decline.
Conclusions:
- The study established reliable methods for measuring intramitochondrial free Mg2+ using fluorescent indicators.
- Mitochondrial matrix Mg2+ concentration is dynamic and sensitive to respiratory activity and external Mg2+ availability.
- These findings provide crucial insights into mitochondrial magnesium homeostasis and its regulation.