Name: inner mitochondrial membrane sensitivity to na+ reveals partially segmented functional coq pools
Uploaded: 2022-07-20T13:15:00
Description: Watch this Scientific Journal Video about Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools at JoVE.com

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...

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>K2HPO4</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>MgCl2</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

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...

Watch this Scientific Journal Video about Inner Mitochondrial Membrane Sensitivity to Na+ Reveals Partially Segmented Functional CoQ Pools at JoVE.com

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

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.

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 ...

This protocol highlights the role of sodium as a second messenger and also shows the existence of partially differentiated ubiquinol pools. This technique demonstrates an extremely simple approach for the study of ubiquinol pools which otherwise requires very specific cell models. This approach may be used for the study of electron transfer in other membranes, particularly of enzymes within lipid drops.Split the samples into four sub-samples of 20 micrograms each and label them A to D.Split samples A and B into two sub-samples of 10 micrograms each and label them as A1, A2, B1, and B2.Prepare C1/C2 buffer as described in the text protocol and preheat to 37 degrees Celsius. In a one milliliter cuvette, add each of the sub-samples to 30 microliters of cytochrome c and 10 microliters of malonate. Add C1/C2 buffer to bring up the volume to 980 microliters in cuvettes A1 and B1 and 979 microliters in A2 and B2.Add 10 microliters of one-molar potassium chloride in cuvettes A1 and A2 and 10 microliters of one-molar sodium chloride in B1 and B2.Add one microliter of one-millimolar rotenone into the cuvettes A2 and B2.Right before the measurement, add 10 microliters of 10-millimolar NADH into all cuvettes.Flip the cuvette three times and place it in the spectrophotometer. An the attached software, click on Measure then Parameters, then go to General and set the measurement parameters at wavelength of 550 nanometers and time at four minutes of reading. Click on OK and Start to begin the experiment.At the end of the measurement, save the slope comprising the linear increase of absorbance by clicking on File and Save As.Split samples C and D into two sub-samples of 10 micrograms each and label them as C1, C2, D1, and D2.Mix each of the sub-samples in a one-milliliter cuvette with 30 microliters of cytochrome c and one microliter of rotenone. Add C1/C2 buffer preheated at 37 degrees Celsius to bring up the volume to 980 microliters in cuvettes C1 and D1 and 970 microliters in C2 and D2.Add 10 microliters of one-molar potassium chloride in cuvettes C1 and C2 and 10 microliters of one-molar sodium chloride in D1 and D2.Add one microliter of one-millimolar antimycin A into the cuvettes C2 and D2 right before the measurement and 10 microliters of one-molar succinate into all cuvettes. Flip the cuvette three times and place it in the spectrophotometer.Perform the measurement and save the slope comprising the linear increase of absorbance at the end of the measurement. This protocol was used to study the effect of sodium ions on the reduction of cytochrome c by mitochondrial membranes upon NADH or succinate addition. Absorbance traces generated at 550 nanometers for samples A and B were corrected for their corresponding inhibition.These traces showed a similar slope, indicating similar NADH-cytochrome c oxidoreductase activity. However, traces for samples C and D were different, showing that succinate-cytochrome c oxidoreductase activity of sample C is higher than that of sample D.In order to normalize the amount of change measured with this technique, other methods can be further performed, such as isolated complex I activity, complex II, or complex III. Using this technique, we are exploring the physiological settings in which sodium may be involved as a second messenger.

Research

JoVE Journal

Biochemistry

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