Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium‐Selective Electrode

António J. Moreno1, Dario L. Santos2, Sílvia Magalhães‐Novais3, Paulo J. Oliveira3

1 DCV – Department of Life Sciences, Universidade de Coimbra, Coimbra, 2 Centre for the Research & Technology of Agro‐Environmental & Biological Sciences (CITAB), Universidade de Trás‐os‐Montes & Alto Douro (UTAD), Vila Real, 3 CNC – Center for Neuroscience and Cell Biology, UC‐Biotech Building, Biocant Park, Universidade de Coimbra, Cantanhede
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 25.5
DOI:  10.1002/0471140856.tx2505s65
Online Posting Date:  August, 2015
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Abstract

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. © 2015 by John Wiley & Sons, Inc.

Keywords: mitochondrial membrane potential; tetraphenylphosphonium cation; selective electrode; isolated mitochondria

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the TPP+‐Selective Electrode
  • Basic Protocol 2: Assembly of the TPP+‐Selective Electrode
  • Basic Protocol 3: Measuring Mitochondrial ΔΨ with the TPP+‐Selective Electrode in Isolated Mitochondrial Fractions
  • Support Protocol 1: Calculating Mitochondrial ΔΨ
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of the TPP+‐Selective Electrode

  Materials
  • High‐molecular‐weight polyvinylchloride (PVC; mol. wt. 200,000)
  • Tetrahydrofurane (THF; Merck)
  • Bis(2‐ethylhexyl) sebacate (sebacic acid; Fluka)
  • Sodium‐tetraphenylborate (Aldrich)
  • Potassium‐tetraphenylborate (Fluka)
  • Tetraphenylphosphonium chloride (TPPCl; Fluka)
  • 15‐ml glass vessel using magnetic stirring
  • Fume hood
  • 10‐ml glass tubes
  • Glass pipets
  • Six glass rings, 2‐cm diameter
  • Large beakers
  • 80‐mm PVC tubing (2‐mm diameter)
  • Scalpel
  • Scissors
  • Small syringe with a long needle
  • Ag/AgCl wire soldered to an appropriate pH meter cable
NOTE: It is preferable to use Aldrich, cat. no. T2,540‐2. Tetraphenylboron from other suppliers may not work properly.

Basic Protocol 2: Assembly of the TPP+‐Selective Electrode

  Materials
  • TPP+‐selective electrode (see protocol 1)
  • Teflon tape
  • Metal clamps
  • Metal rods
  • Aluminum foil
  • Wire Faraday cage
  • pH meter (e.g., Jenway 3520 pH meter or similar)
  • Reference electrode (Ag/AgCl saturated electrode Tacussel, model MI 402 or similar)
  • Paper recorder (Kipp & Zonen, model BD 112)

Basic Protocol 3: Measuring Mitochondrial ΔΨ with the TPP+‐Selective Electrode in Isolated Mitochondrial Fractions

  Materials
  • Assay medium (see recipe)
  • Glutamate/malate 1 M/0.5 M (pH 7.2), optional (see recipe)
  • TPP+‐selective electrodes (as prepared in Basic Protocols protocol 11 and protocol 22)
  • 10 mM TPPCl (see recipe)
  • Isolated mitochondrial fraction (e.g., liver, heart, brain, kidney, etc.)
  • 1 mM FCCP (see recipe)
  • 0.2 mg/ml valinomycin (see recipe)
  • Deionized water
  • Ethanol
  • Magnetic stirrer
  • pH meter (e.g., JENWAY 3520 pH meter)
  • Suitable recorder (e.g., Kipp & Zonen, BD 112)
  • Temperature‐controlled open incubation chamber
  • Circulating water bath
  • Reference electrode (e.g., Ag/AgCl saturated reference electrode ; Tacussel, model MI 402)
NOTE: All stocks are stored at −20°C. Further chemicals are dependent on the type of assay required. Adenosine diphosphate (ADP) phosphorylation cycles are usually performed with the addition of 100 to 500 nmol ADP (see recipe).
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Figures

Videos

Literature Cited

Literature Cited
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