Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium-Selective Electrode.

Mitochondrial bioenergetics is based on the generation of the protonmotive force by the electron transport chain. The protonmotive force is used by mitochondria for different critical aspects of its normal function, ranging from calcium accumulation to the synthesis of ATP. The transmembrane electric potential (ΔΨ) is the major component of the protonmotive force and is also the main responsible for ATP synthesis by mitochondrial ATP synthase. Although several methods can be used to measure the ΔΨ, the use of the tetraphenylphosphonium cation (TPP(+))-selective electrode is still a method of election due to its sensitivity. The method is based on the accumulation of TPP(+) by energized mitochondria, which develop a negative charge in the matrix due to the ejection of protons. This unit describes how to build a custom-made TPP(+)-selective electrode and how to establish the necessary set-up to follow ΔΨ fluctuations in isolated mitochondrial fractions.