Name: the use of the patch-clamp technique to study the thermogenic capacity of mitochondria
Uploaded: 2021-05-03T15:00:00
Description: Watch this Scientific Journal Video about The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria at JoVE.com

I thank Dr. Yuriy Kirichok for the great science I was part of in his lab and the members of the Kirichok lab for the helpful discussions. I also thank Dr. Douglas C. Wallace for providing AAC1 knockout mice. Funding: A.M.B was supported by an American Heart Association Career Development Award 19CDA34630062.

All animal experimental procedures that were performed conform to the National Institutes of Health guidelines and were approved by the University of California Los Angeles Institutional Animal Care and Use Committee (IACUC).
NOTE: The mitochondrial isolation procedure is based on differential centrifugation and varies slightly from tissue to tissue. For example, since brown adipose tissue is extremely rich in lipids, it requires an additional step to separate cell debris and organelles from t...

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This method article aims to present the patch-clamp technique recently applied to mitochondria, a new approach to directly study H+ leak through the IMM responsible for mitochondrial thermogenesis5,6,7,15. This technique is not limited to tissues and can also be used to analyze H+ leak and other conductances of the IMM in different standard human and cell models such as HA...

Mitochondria are famous for being the powerhouse of the cell. Indeed, they are the major source of chemical energy, ATP. What is less known is that mitochondria also generate heat. In fact, every mitochondrion constantly generates the two types of energies (ATP and heat) and a fine balance between the two energy forms defines metabolic cell homeostasis (Figure 1). How mitochondria distribute energy between ATP and heat is certainly the most fundamental question in the field of bioenergetics, although it is still largely unknown. We do know that increasing mitochondrial heat production (called mitochondrial thermogenesis), and consequently reducing ATP production increases energy expenditure and this is one of the best ways to combat metabolic syndrome1.
Mitochondrial thermogenesis originates from H+ leak across the inner mitochondrial membrane (IMM), leading to uncoupling of substrate oxidation and ATP synthesis with consequent production of heat, hence the name &#34;mitochondrial uncoupling&#34;1 (Figure 1). This H+ leak depends on mitochondrial transporters called uncoupling proteins (UCPs). UCP1 was the first UCP identified. It is only expressed in thermogenic tissues, brown fat, and beige fat in which mitochondria are specialized for heat production2,3,4. The identity of UCP in non-adipose tissues such as skeletal muscle, heart, and liver, has remained controversial. Mitochondria in these tissues can have about 25% of the total mitochondrial energy converted into heat, which can significantly impact the physiology of the whole body1. Besides maintaining core body temperature, mitochondrial thermogenesis also prevents diet-induced obesity by reducing calories. In addition, it reduces the production of reactive oxygen species (ROS) by mitochondria to protect cells from oxidative damage1. Thus, mitochondrial thermogenesis is involved in normal aging, age-related degenerative disorders, and other conditions involving oxidative stress, such as ischemia-reperfusion. Therefore, mitochondrial thermogenesis is a powerful regulator of cellular metabolism, and a mechanistic understanding of this fundamental process will promote the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction.
Mitochondrial respiration was the first technique to reveal the crucial role of mitochondrial thermogenesis in cellular metabolism and is still the most popular in the community1. This technique is based on the measurement of oxygen consumption by the mitochondrial electron transport chain (ETC) that increases when mitochondrial H+ leak is activated. This technique, although instrumental, cannot directly study mitochondrial H+ leak across the IMM1, thereby making the precise identification and characterization of the proteins responsible for it difficult, particularly in non-adipose tissues in which heat production is secondary as compared to ATP production. Recently, the development of the patch-clamp technique applied to mitochondria, provided the first direct study of H+ leak across the whole IMM in various tissues5,6,7.
The mitochondrial patch-clamp of the whole IMM was first established in a reproducible way by Kirichok et al.8. They described the first direct measurement of mitochondrial calcium uniporter (MCU) currents in 2004 using mitoplasts from COS-7 cell lines8. Later, the Kirichok lab showed calcium currents from IMMs of mouse9 and Drosophila tissues9. Other labs now routinely use this technique to study the biophysical properties of MCU10,11,12,13,14. Whole IMM patch-clamp analysis of potassium and chloride conductance is also possible and has been mentioned in several papers but has not yet been the main subject of a publication6,7,9. The first measurement of H+ currents across the IMM was reported in 2012 from mouse brown fat mitochondria6, and from mouse beige fat mitochondria in 20177. This current is due to the specific uncoupling protein of thermogenic tissues, UCP16,7. Recent work published in 2019 characterized AAC as the main protein responsible for mitochondrial H+ leak in non-adipose tissues such as the heart and skeletal muscle5.
This unique approach now allows for the direct high-resolution functional analysis of the mitochondrial ion channels and transporters responsible for mitochondrial thermogenesis. To facilitate the expansion of the method and to complement other studies such as mitochondrial respiration, a detailed protocol is described below for measuring the H+ currents carried by UCP1 and AAC. Three important steps are described: 1) mitochondrial isolation from mouse brown fat to analyze UCP1-dependent H+ current and mitochondrial isolation from the heart to analyze AAC-dependent H+ current, 2) preparation of mitoplasts with a French Press for mechanical rupture of the outer mitochondrial membrane (OMM), 3) patch-clamp recordings of UCP1 and AAC-dependent H+ currents across the whole IMM.

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			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
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			<td>60X water immersion objective, numerical aperture 1.20</td>
			<td>Olympus</td>
			<td>UPLSAPO60XW</td>
			<td></td>
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			<td>Axopatch 200B amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
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			<td>Borosilicate glass capillaries</td>
			<td>Sutter Instruments</td>
			<td>BF150-86-10</td>
			<td></td>
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			<td>Digidata 1550B Digitizer</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
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			<td>Faraday cage</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
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			<td>French Press</td>
			<td>Glen Mills</td>
			<td>5500-000011</td>
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			<td>IKA Eurostar PWR CV S1 laboratory overhead stirrer</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Inversed Microscope</td>
			<td>Olympus</td>
			<td>IX71 or IX73</td>
			<td></td>
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			<td>Micro Forge</td>
			<td>(Narishige)</td>
			<td>MF-830</td>
			<td></td>
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			<td>Micromanupulator MPC-385</td>
			<td>Sutter Instruments</td>
			<td>FG-MPC325</td>
			<td></td>
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		<tr>
			<td>Microelectrode holder for agar bridge</td>
			<td>World Precision Instruments</td>
			<td>MEH3F4515</td>
			<td></td>
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			<td>Micropipette Puller</td>
			<td>(Sutter Instruments)</td>
			<td>P97</td>
			<td></td>
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			<td>Mini Cell for French Press</td>
			<td>Glen Mills</td>
			<td>5500-FA-004</td>
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			<td>MIXER IKA 6-2000RPM</td>
			<td>Cole Parmer</td>
			<td>EW-50705-50</td>
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			<td>Objective 100X magnification</td>
			<td>Nikon&#160; lens</td>
			<td>MPlan 100/0.80 ELWD 210/0</td>
			<td></td>
		</tr>
		<tr>
			<td>pClamp 10</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion chamber</td>
			<td>Warner Instruments</td>
			<td>RC-24E</td>
			<td></td>
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			<td>Potter-Elvehjem homogenizer 10 ml</td>
			<td>Wheaton</td>
			<td>358039</td>
			<td></td>
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			<td>Refrigerated centrifuge SORVALL X4R PRO-MD</td>
			<td>Thermo Scientific</td>
			<td>75 009 521</td>
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			<td>Small round glass coverslips: 5 mm diameter, 0.1 mm thickness</td>
			<td>Warner Instruments</td>
			<td>640700</td>
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			<td>Vibration isolation table</td>
			<td>Newport</td>
			<td>VIS3036-SG2-325A</td>
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			<td>Chemicals</td>
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			<td>D-gluconic acid</td>
			<td>Sigma Aldrich</td>
			<td>G1951</td>
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			<td>D-mannitol</td>
			<td>&#160;Sigma Aldrich</td>
			<td>M4125</td>
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			<td>EGTA</td>
			<td>&#160;Sigma Aldrich</td>
			<td>3777</td>
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			<td>HEPES</td>
			<td>&#160;Sigma Aldrich</td>
			<td>H7523</td>
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			<td>KCl</td>
			<td>&#160;Sigma Aldrich</td>
			<td>60128</td>
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			<td>MgCl2</td>
			<td>&#160;Sigma Aldrich</td>
			<td>63068</td>
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			<td>sucrose</td>
			<td>&#160;Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
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			<td>TMA</td>
			<td>&#160;Sigma Aldrich</td>
			<td>331635</td>
			<td></td>
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			<td>TrisBase</td>
			<td>&#160;Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
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			<td>TrisCl</td>
			<td>&#160;Sigma Aldrich</td>
			<td>T3253</td>
			<td></td>
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the use of the patch-clamp technique to study the thermogenic capacity of mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

The development of the patch-clamp methodology applied to mitochondria provided the first direct study of H+ leak through the IMM and the mitochondrial transporters, UCP1 and AAC, which are responsible for it. The electrophysiological analysis of UCP1- and AAC-dependent H+ leaks can provide a first glance of the thermogenic capacity of mitochondria. The results section describes the standard procedures to measure H+ leak via UCP1 and AAC.
UCP1-dependent...

Watch this Scientific Journal Video about The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria at JoVE.com

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...

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