Mitochondrial membrane potential estimated with the correction of probe binding

Area of Science:

• Biophysics
• Mitochondrial Physiology
• Membrane Transport

Background:

• Lipophilic ions are crucial probes for measuring membrane potential.
• Accurate estimation is hindered by probe binding to cellular components.
• A previously developed correction method for probe binding exists.

Purpose of the Study:

• To apply a novel probe binding correction method to determine intact mitochondrial membrane potential.
• To validate the accuracy and consistency of the corrected membrane potential measurements.

Main Methods:

• Utilized a homologous series of lipophilic cations ((Phe)3-P+-(CH2)n-CH3, n=0-4) and tetraphenylphosphonium (TPP+) as probes.
• Applied a previously established correction method for probe binding to de-energized and energized mitochondria.
• Determined probe binding parameters using Langmuir adsorption isotherm at varying probe concentrations.

Main Results:

• Mitochondrial membrane potential was estimated at -147 mV (interior-negative) under specific energized conditions.
• Probe binding exhibited Langmuir isotherm characteristics with distinct high- and low-affinity sites.
• The correction method ensured consistent membrane potential values across different lipophilic probes.

Conclusions:

• The developed correction method accurately determines mitochondrial membrane potential by accounting for probe binding.
• The method's validity is confirmed by consistent results obtained with various probes.
• An expanded method incorporating the Langmuir adsorption isotherm reduces probe concentration dependency.