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SUIT-031 Q ce-pce D074

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SUIT-031 Q ce-pce D074

Description

Ce1;1Dig;1PM;2D;3S;4Rot;5U;6Anoxia.png

Abbreviation: PM+S+Rot

Reference: A to analyse O2 flux and the Q-redox state in the NS- pathway control state on permeabilized cells- SUIT-031

SUIT number: D074_ce1;1Dig;1PM;2D;3S;4Rot;5U;6Anoxia;7Ama

O2k-Application: Q

SUIT-031 Q ce-pce D074 is a protocol to investigate simultaneously oxygen flux and the Q-redox state in the N- and NS-pathway and S-pathway control state in OXPHOS and in the S-pathway control state in the ET state in permeabilized cells. After permeabilization of the plasma membrane, coenzyme Q2 mimetic is titrated since the naturally occurring oQ 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 reactions 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.
The addition of PM leads to a partial reduction of CoQ2 which is reflected in the increase of the Q-signal. ADP oxidizes CoQ2, reflected in the decrease of the Q-signa. Addition of S initiates NS-OXPHOS capacity and results in further reduction of CoQ2 in the OXPHOS state. Rotenone via CI inhibition reduces O2 flux to S-linked OXPHOS capacity and causes oxidation of CoQ2. 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 the full reduction of CoQ2. This step is used as a reference step when calculating the Q redox 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-031 DLP files are currently provided for different applications. For O2 application with ce-pce, choose [[]], for mt, choose SUIT-031. For Q redox state detection with mt, choose SUIT-031 Q mt D072.

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

Communicated by Komlodi T (last update 2021-11-25) 

Representative traces

D074 O2 traces.png 700px

MitoPedia: SUIT

Steps and respiratory states

Ce1;1Dig;1Q2;1PM;2D;3S;4Rot;5U;6Anox;7Ama.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 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).
1PM PML(n) N CI 1Q2;1PM
2D PMP N CI 1Q2;1PM;2D
3S PMSP NS CI&II 1Q2;1PM;2D;3S
  • Respiratory stimulation by simultaneous action of type N substrates & succinate, with convergent electron flow in the NS-pathway for reconstitution of TCA cycle function.
  • OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4Rot SP S CII 1Q2;1PM;2D;3S;4Rot
5U SE N CII 1Q2;1PM;2D;3S;4Rot;5U
6Anox 1Q2;1PM;2D;3S;4Rot;5U;6Anox
  • 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.
7Ama ROX 1Q2;1PM;2D;3S;4Rot;5U;6Anox;7Ama
  • 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).



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Strengths and limitations

  • SUIT-031 Q ce-pce D074 in combination with SUIT-006 Q ce-pce D073 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 a mt-database on comparative mitochondrial physiology.
3. to screen specific defects.
  • Cytochrome c test can be performed in the following protocol: [[ ]].
  • This protocol can be extended with the Complex IV module in the following protocol: [[ ]].
  • To study the effect of CoQ2 on mitochondrial respiration, the following protocol can be used in a parallel experiment: [[ ]].
+ Reasonable duration of the experiment.
- The fully oxidized CoQ2 cannot be detected with rotenone in this protocol. This protocol can be run simultaneously with SUIT-006 Q ce-pce D073 to determine the fully oxidized CoQ2. If it is not applicable, the effect of rotenone can be tested on the Q-signal in a separate experiment using the same sample under the same experimental conditions (same respiration medium, same sample concentration). If rotenone does not have a further effect on the Q signal in the presence of sample and coenzyme Q2, this test can be omitted.
- Omy concentration has to be determined if used. Higher concentrations of Omy may inhibit the ET state.
- 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.
- CIV activity cannot be determined and cytochrome c test cannot be performed together with the Q-Sensor.

Compare SUIT protocols

  • SUIT-006 O2 mt D047 is a coupling-control protocol in the N-pathway for isolated mitochondria, tissue homogenate and cells permeabilized before addition to the O2k- chamber.
  • SUIT-004 is designed to provide a quick assessment of the linear coupling control (L- P- E) with NADH-linked substrates (PM) and the contribution of the S-pathway to the ET state (N, NS, S).
  • SUIT-008 is designed to assess the additivity between the N- and S-pathway in the Q-junction, providing a physiologically relevant estimate of maximum mitochondrial respiratory capacity.


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
1PM Pyruvate (P) and Malate (M)
2D ADP (D)
3S Succinate (S)
4Rot Rotenone (Rot)
5U Carbonyl cyanide m-chlorophenyl hydrazone, CCCP (U) Can be substituted for other uncoupler.
6Anox The O2 concentration in the O2k-chamber can be decreased by N2 or H2 injection to reach faster anoxia, see: Setting the oxygen concentration.
7Ama 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


MitoPedia concepts: SUIT protocol, SUIT A, Find 


MitoPedia methods: Respirometry