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[Erroneous use of lipophilic phosphonic cations for determining mitochondrial membrane potential]
Abstract:
The energy-dependent accumulation of triphenylmethylphosphonium (TPMP+) in rat liver mitochondria at 0 degrees C is less than that at a room temperature only by 30-50%, while the cation penetration rate drastically decreases thus making possible an accurate studying of the transport kinetics. It is shown that K+-diffusion potential accelerates the influx of TPMP+ and decreases the efflux rate in full agreement with the expected behaviour of a penetrating cation diffusing along or against the electrical field. On the contrary, the energization of mitochondria, which has been generally believed to generate a metabolic membrane potential, caused only a stimulation of the influx of TPMP+ without affecting the efflux rate. The TPMP+ efflux half-time was found to be about 3 minutes independently of the energetic state of mitochondria. This independence indicates that the energization is not accompanied by a rise of the membrane potential. The accumulation of TPMP+ by the energized mitochondria must be considered as an active process transporting lipophilic cations against the electrochemical gradient. It follows that the steady state distribution of TPMP+ cannot be a measure of the membrane potential in mitochondria.
Insights
Triphenylmethylphosphonium (TPMP+) accumulation in mitochondria is not solely driven by membrane potential. Energization actively transports TPMP+ against the electrochemical gradient, suggesting it
Area of Science:
- Mitochondrial physiology
- Membrane transport
- Bioenergetics
Background:
- Mitochondrial membrane potential is crucial for cellular energy production.
- Triphenylmethylphosphonium (TPMP+) is a lipophilic cation used to probe mitochondrial membrane potential.
- Previous studies suggested TPMP+ accumulation reflects membrane potential.
Purpose of the Study:
- To investigate the relationship between mitochondrial energization and TPMP+ accumulation.
- To determine if TPMP+ distribution accurately reflects the mitochondrial membrane potential.
- To elucidate the mechanism of TPMP+ transport in energized mitochondria.
Main Methods:
- Studying TPMP+ accumulation and efflux kinetics in rat liver mitochondria at different temperatures (0°C and room temperature).
- Utilizing K+-diffusion potential to assess cation transport dynamics.
- Comparing TPMP+ behavior under energized and non-energized mitochondrial states.
Main Results:
- TPMP+ influx is accelerated by K+-diffusion potential, while efflux is decreased, consistent with passive diffusion.
- Mitochondrial energization stimulates TPMP+ influx but does not affect its efflux rate.
- TPMP+ efflux half-time remains constant (~3 minutes) regardless of mitochondrial energetic state.
- This indicates mitochondrial energization does not increase membrane potential as measured by TPMP+.
Conclusions:
- Mitochondrial energization drives an active process for TPMP+ accumulation against the electrochemical gradient.
- TPMP+ steady-state distribution is not a reliable indicator of mitochondrial membrane potential.
- The findings challenge the conventional use of TPMP+ for quantifying mitochondrial membrane potential.