Name: Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools (Video) | JoVE Journal
Uploaded: 2022-07-20T13:15:00.000+00:00
Duration: 5 min 27 s
Description: Discover a simple assay to study partially segmented functional CoQ pools and sodium's role as a second messenger in mitochondrial electron transfer. This method simplifies investigating ubiquinol pool differentiation without requiring specific cell models.

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5 minutes

We thank Dr. R. Mart&#237;nez-de-Mena, M. M. Mu&#241;oz-Hernandez, A., Dr C. Jimenez and E. R. Mart&#237;nez-Jimenez for technical assistance. This study was supported by MICIN: RTI2018-099357-B-I00 and HFSP (RGP0016/2018). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia, Innovaci&#243;n y Universidades (MCNU) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Figure 2 created with BioRender.com.

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
<ol>
	<li>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.</li>
	<li>Quantify the protein amount of the isolated mitochondria sample by Bradford or Bicinchoninic acid (BCA) methods. In the case of Bradford, add 2 &#181;L of sample into 1 mL of 1x Bradford reagent.</li>
	<li>Split the sample into four subsamples of 20 &#181;g each (namely: A, B, C, D; Figure 2A).</li>
</ol>
2. Measuring CI+CIII activity
NOTE: This part of the protocol uses samples A and B to measure CI+CIII activity (Figure 2B).
<ol>
	<li>Split samples A and B into two subsamples of 10 &#181;g each (namely A1, A2, B1, and B2). Mix each of the subsamples in a 1 mL cuvette with 30 &#181;L of cyt c (10 mg/mL), 10 &#181;L of 100 mM malonate, and add preheated C1/C2 buffer (Table 1) at 37 &#176;C up to 980 &#181;L (979 &#181;L 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.</li>
	<li>Add 10 &#181;L of 1 M KCl in cuvettes A1 and A2, and add 10 &#181;L of 1 M NaCl in cuvettes B1 and B2.</li>
	<li>Add 1 &#181;L of 1 mM rotenone into the cuvette containing subsamples A2 and B2. 
	CAUTION: This step involves the use of the toxic reagent rotenone.</li>
	<li>Right before the measurement, add 10 &#181;L of NADH (10 mM) into all cuvettes. 
	NOTE: The 10 &#181;L is preferably added on the step of the cuvette, so the reaction starts upon mixing.</li>
	<li>Mix the cuvette by carefully flipping it three times. Place it in the absorbance cuvette reader (UV/VISJASCO spectrophotometer).</li>
	<li>Click on Measure &#62; Parameters &#62; 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.</li>
	<li>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.</li>
</ol>
3.&#160;Measuring CII+CIII activity
NOTE: This part of the protocol uses samples C and D to measure CII+CIII activity (Figure 2C).
<ol>
	<li>Split samples C and D into two subsamples of 10 &#181;g each (namely C1, C2, D1, and D2). Mix each of the subsamples in a 1 mL cuvette with 30 &#181;L of cyt c (10 mg/mL), 1 &#181;L of 1 mM rotenone, and add preheated C1/C2 buffer at 37 &#176;C up to 980 &#181;L (970 &#181;L 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.</li>
	<li>Add 10 &#181;L of 1 M KCl in cuvettes C1 and C2, and add 10 &#181;L of 1 M NaCl in cuvettes D1 and D2.</li>
	<li>Add 1 &#181;L 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.</li>
	<li>Right before the measurement, add 10 &#181;L of succinate (1 M) into all cuvettes. 
	NOTE: The 10 &#181;L is preferably added on the step of the cuvette, so the reaction starts upon mixing.</li>
	<li>Mix the cuvette carefully, flipping it three times. Place it in the absorbance cuvette reader (UV/VIS spectrophotometer).</li>
	<li>Click on Measure &#62; Parameters &#62; 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.</li>
	<li>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.</li>
</ol>

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<li>Moe, A., Di Trani, J., Rubinstein, J. L., Brzezinski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-EM+structure+and+kinetics+reveal+electron+transfer+by+2D+diffusion+of+cytochrome+c+in+the+yeast+III-IV+respiratory+supercomplex%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Cryo-EM structure and kinetics reveal electron transfer by 2D diffusion of cytochrome c in the yeast III-IV respiratory supercomplex.</a> Proceedings of the National Academy of Sciences of the United States of America. 118 (11), 2021157118(2021).</li>
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<li>Hernansanz-Agustin, P., Enriquez, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+segmentation+of+CoQ+and+cyt+c+pools+by+respiratory+complex+superassembly%5BTitle%5D)+AND+%22Free+Radical+Biology+and+Medicine%22%5BJournal%5D)">Functional segmentation of CoQ and cyt c pools by respiratory complex superassembly.</a> Free Radical Biology and Medicine. 167, 232-242 (2021).</li>
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<li>den Brave, F., Becker, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Supercomplex+formation+boosts+respiration%5BTitle%5D)+AND+%22EMBO+Reports%22%5BJournal%5D)">Supercomplex formation boosts respiration.</a> EMBO Reports. 21 (12), 51830(2020).</li>
<li>Perez-Mejias, G., Guerra-Castellano, A., Diaz-Quintana, A., Dela Rosa, M. A., Diaz-Moreno, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytochrome+c%3A+Surfing+off+of+the+mitochondrial+membrane+on+the+tops+of+Complexes+III+and+IV%5BTitle%5D)+AND+%22Computational+and+Structural+Biotechnology+Journal%22%5BJournal%5D)">Cytochrome c: Surfing off of the mitochondrial membrane on the tops of Complexes III and IV.</a> Computational and Structural Biotechnology Journal. 17, 654-660 (2019).</li>
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<li>Acin-Perez, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+approach+to+measure+mitochondrial+respiration+in+frozen+biological+samples%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">A novel approach to measure mitochondrial respiration in frozen biological samples.</a> The EMBO Journal. 39 (13), 104073(2020).</li>
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</ol>

The authors declare no conflicts of interest.

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&#39;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...

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 decades2,7,11,12,13,14,15,16,17. 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 (CoQNAD) and another pool dedicated to FAD-dependent enzymes (CoQFAD)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 required1,19,20.
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., CoQNAD), and another dedicated to FAD-linked enzymes (i.e., CoQFAD)1,3,7; an observation that, though it continues to be debated22, has been corroborated independently by several groups7,19. Thus, the superassembly of CI into supercomplexes impacts on the local mobility of CoQ, facilitating its usage by the CIII within the supercomplex1,7,13,14,23,24,25.

<table><tbody><tr><td>Antimycin A</td><td>Sigma-Aldrich</td><td>A8674</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>10775835001</td><td></td></tr><tr><td>Bradford protein assay</td><td>Bio-Rad</td><td>5000001</td><td></td></tr><tr><td>Cytochrome c from equine heart</td><td>Sigma-Aldrich</td><td>C7752</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3786</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Malonic acid</td><td>Sigma-Aldrich</td><td>M1296</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>NADH</td><td>Roche</td><td>10107735001</td><td></td></tr><tr><td>Potassium cyanide</td><td>Sigma-Aldrich</td><td>207810</td><td></td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td></td></tr><tr><td>Spectra Manager software</td><td>JASCO</td><td>version 2</td><td></td></tr><tr><td>Spectrophotometer</td><td>UV/VISJASCO</td><td></td><td></td></tr><tr><td>Succinate</td><td>Sigma-Aldrich</td><td>398055</td><td></td></tr></tbody></table>

inner mitochondrial membrane sensitivity to na+ reveals partially segmented functional coq pools

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

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.

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...

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.

Ubiquinone (CoQ) pools in the inner mitochondrial membrane (IMM) are partially segmented to either complex I or FAD-dependent enzymes. Such subdivision ...

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Discover a simple assay to study partially segmented functional CoQ pools and sodium's role as a second messenger in mitochondrial electron transfer. This method simplifies investigating ubiquinol pool differentiation without requiring specific cell models.

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

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