Description
Abbreviation: CCP mt S(Rot)
Reference: A to analyse the coupling control in isolated mitochondria in S(Rot)- pathway control state- SUIT-006
SUIT number: D071_1mt;1Q;1SRot;2D;3Omy;4U;5Anoxia
O2k-Application: Q
SUIT-006 Q mt D071 is a coupling-control protocol for simultaneous measurement of O2 flux and the Q-redox state in isolated mitochondria or permeabilized cells (which are permeabilized before adding them into the O2k-chamber). Different coupling control states are assessed (L(n) - P - E or L(n) - P-L(Omy)- E) at the succinate-pathway control state.
After the addition of mitochondria in the absence of fuel substrates and ADP, Rox with endogenous substrates (Ren) is detected due to oxidation of endogenous substrates remaining after mitochondrial isolation.
Directly after the sample addition, coenzyme Q2 mimetic is titrated. CoQ2 reacts both with the mitochondrial complexes at the Q-binding site (CI, CII, and CIII) and with the detecting electrode of the Q-Sensor. Application of the lowest possible CoQ2 concentration is recommended to avoid any side reactions on the ETS. Our study shows that 1 µM CoQ2 was sufficient to detect the Q-redox change without an influence on respiration.
Rotenone, an inhibitor of Complex I, avoids oxaloacetate accumulation which could inhibit succinate dehydrogenase. Furthermore, rotenone is needed to inhibit mitochondrial oxidation of residual endogenous substrates, allowing to detect the fully oxidized CoQ2 in the presence of sample and the coenzyme Q2 mimetic for calculating the reduced Q fraction.
Succinate as a substrate of Complex II is oxidized to fumarate and supports electron transfer through CII to Q. Succinate with rotenone supports S-linked LEAK respiration and leads to reduction of CoQ2 which is reflected in the increase of the Q-signal.
ADP titration in saturating concentration initiates the OXPHOS state, and increase in respiration is accompanied by slight oxidization of the Q-junction, reflected in a decrease of the Q-signal.
The use of oligomycin is optional, however, it provides important information when residual and endogenous adenylates are present (which may happen if ATPases are active in the sample). This situation may lead to overestimated LEAK respiration measured in the absence of adenylates - L(n). Therefore, oligomycin can be used to verify whether this occurs and obtain the LEAK state appropriately. Since higher concentrations of Omy can decrease the ET state induced upon the addition of uncoupler, the required concentration of Omy has to be assessed by stepwise Omy titration.
The titration of uncoupler such as CCCP leads to the ET state, and CoQ2 may be further oxidized in parallel to increase in respiration, however, using mouse cardiac mitochondria (see figures) U did not influence either O2 flux or the Q redox state which means that the respiration is not limited by the phosphorylation system in this sample type.
Anoxia is reached when the mitochondria fully consume the oxygen in the O2k-chambers. In the absence of O2, the ETS upstream of CIV is reduced and thus leads to full reduction of CoQ2. This step is used as a reference step when calculating the reduced Q fraction. At the end of the protocol, the CIII inhibitor antimycin A can be added to check its effect on the fully reduced CoQ2 under anoxia.
In the DatLab software, SUIT-006 DLP files are currently provided with succinate as a substrate. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created.
On how to measure the Q-redox state, see: MiPNet24.12 NextGen-O2k: Q-Module in preparation
Communicated by Komlodi T (last update 2021-02-01)
Representative traces
Steps and respiratory states
Step | State | Pathway | Q-junction | Comment - Events (E) and Marks (M) |
---|---|---|---|---|
1Q2 | REN | 1Q2
| ||
1Rot | ROX | 1Rot
| ||
1S | SL | S | CII | 1Q2;1Rot;1S
|
2D | SP | S | CII | 1Q2;1Rot;1S;2D
|
(3Omy) | SL(Omy) | S | CII | 1Q2;1Rot;1S;2D;(3Omy)
|
4U | SE | S | CII | 1Q2;1Rot;1S;2D;(3Omy);4U
|
5Anox | S | CII | 1Q2;1Rot;1S;2D;(3Omy);4U;5Anox
| |
6Ama | ROX | 1Q2;1Rot;1S;2D;(3Omy);4U;5Anox;6Ama
|
- Bioblast links: SUIT protocols - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
- Coupling control
- Pathway control
- Main fuel substrates
- » Glutamate, G
- » Glycerophosphate, Gp
- » Malate, M
- » Octanoylcarnitine, Oct
- » Pyruvate, P
- » Succinate, S
- Main fuel substrates
- Glossary
Strengths and limitations
- + Reasonable duration of the experiment.
- - This protocol cannot be extended with the Complex IV module.
- - Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors, if used.
Compare SUIT protocols
Chemicals and syringes
Step | Chemical(s) and link(s) | Comments |
---|---|---|
1Rot | Rotenone (Rot) | This step is essential to reach the fully oxidized CoQ2 needed to calculate the reduced Q fraction. |
1S | Succinate (S) | |
2D | ADP (D) | |
(3Omy) | Oligomycin (Omy) | This step can be skipped. |
4U | Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) | Can be substituted for other uncoupler. |
5Anox | The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration. | |
6Ama | Antimycin A (Ama) | This step can be omitted. |
- Suggested stock concentrations are shown in the specific DL-Protocol.
References
Year | Reference | Organism | Tissue;cell | |
---|---|---|---|---|
MiPNet24.12 NextGen-O2k: Q-Module | 2021-10-29 | NextGen-O2k: Q-Module manual | ||
Komlodi 2021 BEC Q | 2021 | Komlódi T, Cardoso LHD, Doerrier C, Moore AL, Rich PR, Gnaiger E (2021) Coupling and pathway control of coenzyme Q redox state and respiration in isolated mitochondria. Bioenerg Commun 2021.3. https://doi.org/10.26124/bec:2021-0003 | Mouse | Heart Nervous system |
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