How do you assess mitochondrial membrane potential?
Mitochondrial function, a key indicator of cell health, can be assessed by monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in HepG2 cells.
How do you measure a proton leak?
The kinetics of mitochondrial proton leak were measured by sequentially inhibiting respiration that drives the proton leak and plotting respiration rates against their corresponding membrane potentials. Measurements were performed with 1.5 mg/ml of liver mitochondria in KHE buffer.
Do mitochondrial membranes leak protons?
The total proton leak of a mitochondrion can be thought of as the sum of two processes: basal leak, which is unregulated, and inducible leak, which is catalysed by specific mitochondrial inner membrane proteins and can be inhibited and activated.
What is the mitochondrial proton leak?
Proton leak can also be defined as the dissipation of ΔP in the presence of ATP synthase inhibitor Oligomycin in both isolated mitochondria and intact cells [5]. Proton leak is the principal, but not the only, mechanism that incompletely couples substrate oxygen to ATP generation (Fig. 20.1).
How is mitochondrial damage measured?
A very common approach to address mitochondrial bioenergetic dysfunction is to measure the expression, concentration or maximum activity of a few candidate electron transport complexes or metabolic enzymes, such as complex I, complex IV or tricarboxylic acid-cycle enzymes.
How do we measure membrane potential?
The membrane potential is measured using a reference electrode placed in the extracellular solution and a recording electrode placed in the cell soma. The membrane potential is the difference in voltage between these two regions.
What is leak respiration?
LEAK respiration corresponds to resting, non-phosphorylating electron transfer with a shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling.
How does Oligomycin affect electron transport chain?
Oligomycin A is an inhibitor of ATP synthase. The inhibition of ATP synthesis by oligomycin A will significantly reduce electron flow through the electron transport chain; however, electron flow is not stopped completely due to a process known as proton leak or mitochondrial uncoupling.
How does DNP create leaks in the mitochondrial membrane?
When the drug dinitrophenol (DNP) is added to mitochondria, the inner membrane becomes permeable to protons (H+). H+ ions that are pumped to one side of the membrane flow back freely, and therefore no energy to drive ATP synthesis can be stored across the membrane.
Why does a proton leak in the mitochondria generate warmth?
In the presence of thermogenin, oxidation and proton pumping continue at high rates but with low rates of ATP synthesis. So Thermogenin enhances mitochondrial proton leak so there would be heat.”
Can you measure mitochondria?
The efficiency of oxidative phosphorylation can be measured in isolated mitochondria by high-resolution respirometry [30]. The general concept underlying this method is to measure the amount of oxygen consumed for a given amount of ATP synthesized by the mitochondria.