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SUIT-006 Q ce-pce D073

From Bioblast


high-resolution terminology - matching measurements at high-resolution


SUIT-006 Q ce-pce D073

Description

Ce1;1Dig;1Q2;1Rot;1S;2D;(3Omy);4U;5Anox;6Ama.png

Abbreviation: CCP ce-pce S(Rot)

Reference: A Coupling control protocol to analyze mitochondrial O2 consumption and the redox state of the Q-pool in S(Rot)-pathway control state in permeabilized cells- SUIT-006

SUIT number: D073_ce1;1Dig;1Q2;1Rot;1S;2D;(3Omy);4U;5Anox;6Ama.png

O2k-Application: Q


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry 




SUIT-006 Q ce-pce D073 is a coupling-control protocol for simultaneous measurement of O2 flux and the Q-redox state in permeabilized cells. Different coupling control states are assessed (L(n) - P - E or L- P-L(Omy)- E) at the succinate-pathway control state.
After permeabilization of the plasma membrane, coenzyme Q2 mimetic is titrated since the naturally occurring CoQ is trapped in the mitochondrial inner membrane. 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 reaction on the ETS caused by the mimetics. Our study shows that 1 µM CoQ2 was sufficient to detect the Q-redox change without an influence on respiration. Importantly, CoQ2 mimetic should be titrated after permeabilization of the plasma membrane owing to the extramitochondrial Q-pools in the cell.

Rotenone, an inhibitor of Complex I, avoids oxaloacetate inhibition towards succinate dehydrogenase. Furthermore, rotenone is needed to detect the fully oxidized CoQ2 in the presence of sample and the coenzyme Q2 mimetic but in the absence of ADP and succinate 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 an almost full reduction of CoQ2 which is reflected in the increase of the Q-signal.
ADP slightly oxidizes 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 the Omy titration test.

The uncoupler CCCP oxidizes CoQ2, 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 was reached when the mitochondria consumed 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 for the categories N(PM) and S. For using this protocol with other substrate/inhibitor combinations, a personalized DLPU can be created. For O2 application with ce-pce, choose SUIT-006 O2 ce-pce D029 to create the DLPU, for mt, choose SUIT-006 O2 mt D047. For O2 and H2O2 measurements (AmR), choose SUIT-006 AmR mt D048, and for O2 and membrane potential measurements, choose SUIT-006 Fluo mt D034, for ATP measurement (MgG) choose SUIT-006 MgG mt D055, and for Q redox state detection with mt, choose SUIT-006 Q mt D071.

How to measure the Q redox state, see: MiPNet24.12 NextGen-O2k: Q-Module in preparation

Communicated by Komlodi T and Cardoso LHD (last update 2023-12-18)

Representative traces

SUIT-006 Q ce-pce D073 traces O2.png SUIT-006 Q ce-pce D073 traces Q.png


MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1Q2;1Rot;1S;2D;(3Omy);4U;5Anox;6Ama.png

Step State Pathway Q-junction Comment - Events (E) and Marks (M)
ce1 ROUTINE ce1
  • ROUTINE respiration in the physiological coupling state R. Externally added permeable substrates could contribute to this respiratory state.
1Dig REN ce1;1Dig
  • Optimum effective digitonin concentration for complete plasma membrane permeabilization.
Step State Pathway Q-junction Comment - Events (E) and Marks (M)
1Q2 REN 1Q2
  • To detect the redox state of the Q-pool a mimetic coenzyme Q2 is applied in suspension, which reacts both with the detecting electrode and the biological sites (CI, CII and CIII).
  • Respiration in the REN state is due to the presence of residual endogenous substrates in the sample preparation.
  • In the absence of endogenous substrates, this step can be used for calibration of fully oxidized Q.
1Rot ROX 1Rot
  • Rotenone is required to detect the fully oxidized Q-pool in the presence of isolated mitochondria (or permeabilized cells) and coenzyme Q2. Rotenone can inhibit the respiration supported by endogenous substrates that remained after the isolation procedure.
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)
  • Omy addition is skipped in SUIT-006 O2 mt D022
  • Succinate, S ( type S-pathway to Q).
  • Non-phosphorylating resting state (LEAK state); LEAK-respiration, L(Omy), after blocking the ATP synthase with oligomycin.
4U SE S CII 1Q2;1Rot;1S;2D;(3Omy);4U
5Anox S CII 1Q2;1Rot;1S;2D;(3Omy);4U;5Anox
  • Anoxia is a crucial step to detect the fully reduced Q-junction in the presence of coenzyme Q2 after the biological sample has consumed the O2 in the O2k-chamber for calculation of the Q redox state.
6Ama ROX 1Q2;1Rot;1S;2D;(3Omy);4U;5Anox;6Ama
  • Rox is the residual oxygen consumption in the ROX state, due to oxidative side reactions, estimated after addition of antimycin A (inhibitor of CIII). Rox is subtracted from oxygen flux as a baseline for all respiratory states, to obtain mitochondrial respiration (mt).
  • Since the previous step was anoxia, it is essential to reoxygenate the chamber before or after antimycin A titration to be able to measure Rox.


Questions.jpg


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Strengths and limitations

  • SUIT-006 Q ce-pce D073 in combination with SUIT-031 Q ce-pce D074 provides a common reference for comparison of respiratory control in a large variety of species, tissues and cell types. Both SUIT protocols provide a mitochondrial mapping which allows:
1. to obtain reference values.
2. to evaluate mitochondrial physiological diversity, generating an mt-database on comparative mitochondrial physiology.
3. to screen specific defects.
+ Reasonable duration of the experiment.
- Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state.
- CIV activity cannot be determined and cytochrome c test cannot be performed together with the Q-Sensor.
- Careful washing is required after the experiment to avoid carry-over of uncoupler and inhibitors.
- The concentration of the oxidized and reduced Q fraction cannot be determined.
  • Cytochrome c test can be performed in the following protocols after permeabilization of the plasma membrane with digitonin:SUIT-006 O2 mt D022.
  • This protocol can be extended with the Complex IV module in the following protocols after permeabilization of the plasma membrane with digitonin: SUIT-006 O2 mt D022.
  • To study the effect of coenzyme Q2 on mitochondrial respiration, the following protocol can be used in a parallel experiment after permeabilization of the plasma membrane with digitonin: SUIT-006 O2 mt D022.


Compare SUIT protocols

  • SUIT-006 O2 ce-pce D029: Coupling-control protocol for permeabilized cells, can be used as a quality control with cytochrome c titration.
  • SUIT-031 Q ce-pce D074: SUIT protocol for Q with N-, NS- and S-pathway, cross-calibrated with SUIT-006.


Chemicals and syringes

Step Chemical(s) and link(s) Comments
1Dig Digitonin (Dig)
Step Chemical(s) and link(s) Comments
1Q2 Coenzyme Q2 (Q)
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

 YearReferenceOrganismTissue;cell
MiPNet24.12 NextGen-O2k: Q-Module2021-10-29
O2k-Manual
NextGen-O2k: Q-Module manual
Komlodi 2021 BEC Q2021Komlódi 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:2021-0003MouseHeart
Nervous system