Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools

Podsumowanie

This protocol describes a comparative assay, using mitochondrial complex activities CI+CIII and CII+CIII in the presence or absence of Na⁺, to study the existence of partially segmented functional CoQ pools.

Streszczenie

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision can be easily assessed by a comparative assay using NADH or succinate as electron donors in frozen-thawed mitochondria, in which cytochrome c (cyt c) reduction is measured. The assay relies on the effect of Na⁺ on the IMM, decreasing its fluidity. Here, we present a protocol to measure NADH-cyt c oxidoreductase activity and succinate-cyt c oxidoreductase activities in the presence of NaCl or KCl. The reactions, which rely on the mixture of reagents in a cuvette in a stepwise manner, are measured spectrophotometrically during 4 min in the presence of Na⁺ or K⁺. The same mixture is performed in parallel in the presence of the specific enzyme inhibitors in order to subtract the unspecific change in absorbance. NADH-cyt c oxidoreductase activity does not decrease in the presence of any of these cations. However, succinate-cyt c oxidoreductase activity decreases in the presence of NaCl. This simple experiment highlights: 1) the effect of Na⁺ in decreasing IMM fluidity and CoQ transfer; 2) that supercomplex I+III₂ protects ubiquinone (CoQ) transfer from being affected by lowering IMM fluidity; 3) that CoQ transfer between CI and CIII is functionally different from CoQ transfer between CII and CIII. These facts support the existence of functionally differentiated CoQ pools in the IMM and show that they can be regulated by the changing Na⁺ environment of mitochondria.

Wprowadzenie

Mitochondrial oxidative phosphorylation system (OXPHOS) is the main pathway driving adenosine triphosphate (ATP) synthesis, reactive oxygen species (ROS) production, and consumption of reducing equivalents, such as nicotinamide adenine dinucleotide (NADH) or succinate, by mitochondria. OXPHOS system is composed of five protein complexes: Complex I (CI) oxidizes NADH and reduces CoQ into ubiquinol (CoQH₂). Complex II (CII) oxidizes succinate into fumarate and reduces CoQ into CoQH₂. Complex III (CIII) oxidizes CoQH₂ back into CoQ, reducing cytochrome c (cyt c). Finally, complex IV (CIV) oxidizes cyt c and reduces oxygen to water. This oxidoreduction chain, the so-called electron transport chain (mETC), is coupled to pumping of H⁺ across the IMM, which creates an electrochemical gradient used by complex V (CV) to phosphorylate adenosine diphosphate (ADP) into ATP.

mETC complexes can either be alone in the IMM or assemble into quaternary structures called supercomplexes. CIV can assemble with CIII, forming the III₂+IV or Q-respirasome (as it is able to respire in the presence of CoQH₂)^1,2,3 or forming homodimers or homooligomers⁴. CIII can interact with CI, forming the supercomplex I+III₂⁵. Finally, CI is also able to interact with the Q-respirasome, building the I+III₂+IV or N-respirasome (as it can respire consuming NADH)^1,6,7,8,9,10.

CoQ and cyt c are mobile electron carriers in charge of transferring electrons from CI/CII to CIII, and from CIII to CIV, respectively. Whether or not supercomplexes impose a functional local restriction for these carriers has been a matter of intense debate through the last two decades^{2,7,11,12,13,14,15,16,17}. However, several independent groups have demonstrated that CoQ and cyt c can be functionally segmented into pools in the IMM. In respect of the CoQ, it can be functionally segmented into a specific CoQ pool for CI (CoQ^NAD) and another pool dedicated to FAD-dependent enzymes (CoQ^FAD)^1,7,12,18,19. However, in order to differentiate the existence of partially segmented functional CoQ pools, the overexpression of the alternative oxidase (AOX) and the generation of specific mtDNA mutants, which can assemble CI in the absence of CIII, were required^1,19,20.

The mechanism of reactive oxygen species (ROS) production during hypoxia was unknown until recently. Upon acute hypoxia, CI undergoes the active/deactive (A/D) transition, which involves the decrease in its H⁺ pumping NADH-CoQ oxidoreductase activity. Such a decrease in H⁺ pumping acidifies the mitochondrial matrix and partially dissolves the calcium-phosphate precipitates in the mitochondrial matrix, releasing soluble Ca²⁺. This increase in soluble Ca²⁺ activates the Na⁺/Ca²⁺ exchanger (NCLX), which extrudes Ca²⁺ in exchange for Na⁺. Mitochondrial Na⁺ increase interacts with phospholipids in the inner side of the IMM, decreasing its fluidity and CoQ transfer between CII and CIII, finally producing superoxide anion, a redox signal²¹. Interestingly, CoQ transfer was only diminished between CII and CIII, but not between CI and CIII, highlighting that 1) Na⁺ was able to modulate only one of the existing CoQ pools in the mitochondria; 2) there exists functionally differentiated CoQ pools in the IMM. Thus, a widely used protocol for the study of mitochondrial enzyme activities can be used to assess the existence of the mentioned CoQ pools.

The current protocol is based on the measurement of the reduction of oxidized cyt c, the substrate of CIII, by absorbance in the presence of succinate (i.e., CII substrate) or NADH (i.e., CI substrate). The same sample is divided into two, one of which will be treated with KCl, and the other one with the same concentration of NaCl. In this way, given that Na⁺ decreases IMM fluidity, if CoQ existed in a unique pool in the IMM, both CI+CIII and CII+CIII would decrease in the presence of Na⁺. However, if CoQ existed in partially segmented functional CoQ pools, the effect of Na⁺ would mostly (or only) be evident on the CII+CIII activity, but not on the CI+CIII. As recently published²¹, Na⁺ only affects the CoQ transfer between CII and CIII (Figure 1C,D), but not between CI and CIII (Figure 1A,B).

This protocol, together with a panoply of techniques, has been used to confirm the existence of partially segmented functional CoQ pools in the IMM, one dedicated to CI (i.e., CoQ^NAD), and another dedicated to FAD-linked enzymes (i.e., CoQ^FAD)^1,3,7; an observation that, though it continues to be debated²², has been corroborated independently by several groups^7,19. Thus, the superassembly of CI into supercomplexes impacts on the local mobility of CoQ, facilitating its usage by the CIII within the supercomplex^{1,7,13,14,23,24,25}.

Protokół

All animal experiments were performed following the Guide for the Care and Use of Laboratory Animals and were approved by the institutional ethics committee of the Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Spain, in accordance with the European Union Directive of 22 September 2010 (2010/63/UE) and with the Spanish Royal Decree of 1 February 2013 (53/2013). All efforts were made to minimize the number of animals used and their suffering.

NOTE: This comparative assay to study the segmentation of mitochondrial CoQ pools is described as follows:

1. Protein quantification

1. Freeze and thaw the isolated mitochondria²⁶ from a wild-type mouse liver three times (i.e., mitochondrial membranes) before experimentation to make the organelles permeable to the reaction substrates.
2. Quantify the protein amount of the isolated mitochondria sample by Bradford or Bicinchoninic acid (BCA) methods. In the case of Bradford, add 2 µL of sample into 1 mL of 1x Bradford reagent.
3. Split the sample into four subsamples of 20 µg each (namely: A, B, C, D; Figure 2A).

2. Measuring CI+CIII activity

NOTE: This part of the protocol uses samples A and B to measure CI+CIII activity (Figure 2B).

1. Split samples A and B into two subsamples of 10 µg each (namely A1, A2, B1, and B2). Mix each of the subsamples in a 1 mL cuvette with 30 µL of cyt c (10 mg/mL), 10 µL of 100 mM malonate, and add preheated C1/C2 buffer (Table 1) at 37 °C up to 980 µL (979 µL for cuvettes A2 and B2).
   CAUTION: This step involves the use of the toxic reagents malonate and potassium cyanide.
   NOTE: cyt c (10 mg/mL) must be prepared fresh by mixing 10 mg of cyt c in 1 mL of 10 mM K₂HPO₄ solution, pH adjusted to 7.2, and it must be maintained in ice throughout the experiment.
2. Add 10 µL of 1 M KCl in cuvettes A1 and A2, and add 10 µL of 1 M NaCl in cuvettes B1 and B2.
3. Add 1 µL of 1 mM rotenone into the cuvette containing subsamples A2 and B2.
   CAUTION: This step involves the use of the toxic reagent rotenone.
4. Right before the measurement, add 10 µL of NADH (10 mM) into all cuvettes.
   NOTE: The 10 µL is preferably added on the step of the cuvette, so the reaction starts upon mixing.
5. Mix the cuvette by carefully flipping it three times. Place it in the absorbance cuvette reader (UV/VISJASCO spectrophotometer).
6. Click on Measure > Parameters > General and set the measurement parameters at Wavelength: 550 nm, and Time: 4 min of reading; press Accept and Start buttons to begin the experiment.
7. At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As. The slope can also be collected manually.

3. Measuring CII+CIII activity

NOTE: This part of the protocol uses samples C and D to measure CII+CIII activity (Figure 2C).

1. Split samples C and D into two subsamples of 10 µg each (namely C1, C2, D1, and D2). Mix each of the subsamples in a 1 mL cuvette with 30 µL of cyt c (10 mg/mL), 1 µL of 1 mM rotenone, and add preheated C1/C2 buffer at 37 °C up to 980 µL (970 µL for cuvettes C2 and D2).
   CAUTION: This step involves the use of the toxic reagents potassium cyanide and rotenone.
   NOTE: cyt c (10 mg/mL) must be prepared fresh by mixing 10 mg of cyt c in 1 mL of 10 mM K₂HPO₄ solution, pH adjusted to 7.2, and it must be maintained in ice throughout the experiment.
2. Add 10 µL of 1 M KCl in cuvettes C1 and C2, and add 10 µL of 1 M NaCl in cuvettes D1 and D2.
3. Add 1 µL of 1 mM antimycin A into the cuvette containing subsamples C2 and D2.
   CAUTION: This step involves the use of the toxic reagent antimycin A.
4. Right before the measurement, add 10 µL of succinate (1 M) into all cuvettes.
   NOTE: The 10 µL is preferably added on the step of the cuvette, so the reaction starts upon mixing.
5. Mix the cuvette carefully, flipping it three times. Place it in the absorbance cuvette reader (UV/VIS spectrophotometer).
6. Click on Measure > Parameters > General and set the measurement parameters at Wavelength: 550 nm, and Time: 4 min of reading; press Accept and Start buttons to begin the experiment.
7. At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As. The slope can also be collected manually.

Wyniki

Typical results from this protocol are represented below (Figure 3). As reduced cyt c absorbance locates at 550 nm, all uninhibited subsamples must show an increase in the absorbance at 550 nm. Inhibited subsamples ideally show a flat-line or slightly increasing slope (Figure 3). Slopes from inhibited subsamples are to be subtracted from uninhibited subsamples.

Samples A and B, both corrected by their correspondent inhibition and whic...

Dyskusje

Though this protocol represents a very straightforward procedure to identify the existence of the partially segmented CoQ pools, there are a few critical steps to take into account. Substrates (i.e., NADH or succinate) are preferably added last since autooxidation of these compounds may occur. Cuvette's flipping must be careful in order to avoid the formation of bubbles which may interfere with the reading.

In addition, the present technique presents a few limitations which are worth menti...

Materiały

Name	Company	Catalog Number	Comments
Antimycin A	Sigma-Aldrich	A8674
Bovine Serum Albumin (BSA)	Sigma-Aldrich	10775835001
Bradford protein assay	Bio-Rad	5000001
Cytochrome c from equine heart	Sigma-Aldrich	C7752
K2HPO4	Sigma-Aldrich	P3786
KCl	Sigma-Aldrich	P3911
Malonic acid	Sigma-Aldrich	M1296
MgCl2	Sigma-Aldrich	M8266
NaCl	Sigma-Aldrich	S9888
NADH	Roche	10107735001
Potassium cyanide	Sigma-Aldrich	207810
Rotenone	Sigma-Aldrich	R8875
Spectra Manager software	JASCO	version 2
Spectrophotometer	UV/VISJASCO
Succinate	Sigma-Aldrich	398055