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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The author declares no competing interests.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.1% gelatin</td>
			<td>Millipore</td>
			<td>ES-006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>60X water immersion objective, numerical aperture 1.20</td>
			<td>Olympus</td>
			<td>UPLSAPO60XW</td>
			<td></td>
		</tr>
		<tr>
			<td>Axopatch 200B amplifier</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Sutter Instruments</td>
			<td>BF150-86-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Digidata 1550B Digitizer</td>
			<td>Molecular Devices</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>French Press</td>
			<td>Glen Mills</td>
			<td>5500-000011</td>
			<td></td>
		</tr>
		<tr>
			<td>IKA Eurostar PWR CV S1 laboratory overhead stirrer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inversed Microscope</td>
			<td>Olympus</td>
			<td>IX71 or IX73</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Forge</td>
			<td>(Narishige)</td>
			<td>MF-830</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanupulator MPC-385</td>
			<td>Sutter Instruments</td>
			<td>FG-MPC325</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode holder for agar bridge</td>
			<td>World Precision Instruments</td>
			<td>MEH3F4515</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette Puller</td>
			<td>(Sutter Instruments)</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Cell for French Press</td>
			<td>Glen Mills</td>
			<td>5500-FA-004</td>
			<td></td>
		</tr>
		<tr>
			<td>MIXER IKA 6-2000RPM</td>
			<td>Cole Parmer</td>
			<td>EW-50705-50</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Potter-Elvehjem homogenizer 10 ml</td>
			<td>Wheaton</td>
			<td>358039</td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge SORVALL X4R PRO-MD</td>
			<td>Thermo Scientific</td>
			<td>75 009 521</td>
			<td></td>
		</tr>
		<tr>
			<td>Small round glass coverslips: 5 mm diameter, 0.1 mm thickness</td>
			<td>Warner Instruments</td>
			<td>640700</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibration isolation table</td>
			<td>Newport</td>
			<td>VIS3036-SG2-325A</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-gluconic acid</td>
			<td>Sigma Aldrich</td>
			<td>G1951</td>
			<td></td>
		</tr>
		<tr>
			<td>D-mannitol</td>
			<td>&#160;Sigma Aldrich</td>
			<td>M4125</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>&#160;Sigma Aldrich</td>
			<td>3777</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>&#160;Sigma Aldrich</td>
			<td>H7523</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>&#160;Sigma Aldrich</td>
			<td>60128</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>&#160;Sigma Aldrich</td>
			<td>63068</td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>&#160;Sigma Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>TMA</td>
			<td>&#160;Sigma Aldrich</td>
			<td>331635</td>
			<td></td>
		</tr>
		<tr>
			<td>TrisBase</td>
			<td>&#160;Sigma Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>TrisCl</td>
			<td>&#160;Sigma Aldrich</td>
			<td>T3253</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 ...

This protocol describes how the patch clamp technique can be used to study the thermogenic capacity of mitochondria by directly measuring mitochondrial proton leak across the inner mitochondrial membrane. The direct measurement of the proton current through the inner mitochondrial membrane helps identify and precisely characterize the molecular mechanisms responsible for mitochondrial thermogenesis. This technique allows not only to measure the proton current across the inner mitochondrial membrane, but also to study other conductances important for mitochondrial function, such as calcium or metabolites like adenine nucleotides.Position the euthanized mouse on its back and spray alcohol to clean and wet the hair. Then, make a two-centimeter incision on the thorax. After grasping the skin with tweezers, dissect the heart from the animal's chest and rinse it to remove all the blood into a 10-milliliter beaker with five milliliters of cold isolation solution.Once the heart has been cleared of traces of blood, transfer it to another 10-milliliter beaker containing five milliliters of cold isolation buffer and chop it up into thin pieces. Then, transfer it to an ice-chilled 10-milliliter glass homogenizer. Use an overhead stirrer to homogenize the precut tissue on ice with six gentle strokes at a controlled speed of 275 rotations per minute.Transfer the homogenate to a 15-milliliter ice-cold conical tube and centrifuge it at 700 times G for 10 minutes at four degrees Celsius to pellet the nuclei and unbroken cells. Collect the supernatant in a fresh 15-milliliter tube and place it on ice. Centrifuge the supernatant at 8, 500 times G for 10 minutes at four degrees Celsius to obtain a pellet containing the mitochondria.Resuspend the mitochondrial pellet in 3.8 milliliters of ice-cold hypertonic mannitol buffer and incubate the mitochondrial suspension on ice for 10 to 15 minutes. Fill the mitochondrial hypertonic mannitol suspension into a refrigerated mini-pressure cell of the French press. Then, place the cell onto the French press.Then, select the low mode of the French press and compress the suspension through the mini-pressure cell at 110 on the dial for the brown fat mitochondria, and at 140 for the heart mitochondria, ensuring that the suspension comes out of the mini-pressure cell at a rate of about one drop per second. Collect the drops in a 15-milliliter ice-chilled conical tube. Centrifuge the suspension at 10, 500 times G for 10 minutes at four degrees Celsius.Resuspend the mitoplasts pellet in 0.5 to 2 milliliters of ice-cold hypertonic potassium chloride buffer and store the suspension on ice. Pull the borosilicate glass filaments on the day of recording using a micropipette puller, setting a program on the puller used for generating pipettes with a high degree of reproducibility. Insert one glass filament within the puller and pull to obtain almost two identical patch pipettes from one borosilicate filament.Adjust the program when pipettes become inconsistent between pulling cycles due to aging of the heating box filament of the puller. Position the pipette inside the pipette polisher and place the tip near the filament under 100X magnification to fire polish it. Press the foot pedal several times to heat the filament without clogging or damaging the tip curve.Polish until the pipettes have a resistance between 25 and 35 megaohms are obtained when filled with TMA-based pipette solution. Pre-incubate cover slips with 0.1%gelatin to reduce the mitoplast adhesion and rinse them with the potassium chloride bath solution before depositing the mitoplast suspension. Prepare a raw dilution by mixing approximately 35 microliters of the concentrated mitoplast suspension with 500 microliters of the potassium chloride bath solution and place it on the cover slips previously placed in a well of a four-well plate.Incubate on ice for 15 to 20 minutes for mitoplasts to sediment down the cover slip. Fill the bath chamber completely with approximately 50 microliters of the potassium chloride bath solution and transfer a cover slip with mitoplasts within the chamber using thin microdissection tweezers with a bent tip. Arrange the cover slip at the bottom of the chamber without perfusing the chamber to keep the mitoplasts stable on the cover slip.Choose an individual non-adhesive mitoplast by scanning the cover slip under the microscope with a 60X objective. Load the pipette with the pipette solution and place it in the pipette holder. Bring the pipette into the bath solution with a micromanipulator and move it just above the selected mitoplast to get close to the IMM.Hold the membrane potential at zero millivolts and apply 10 millivolt pulses using the membrane test command in the amplifier program. Apply a slight negative pressure to quickly create a gigaseal with the IMM. Raise the pipette with the mitoplast attached to keep them away from the cover slip to avoid the seal breakage due to pipette drift during the experiment.Compensate the stray capacitance transients with the membrane test command in the amplifier program before testing the whole-mitoplast configuration to obtain a correct capacitance measurement for the mitoplast membrane after the break-in. Apply short-duration voltage pulses with the amplifier program to rupture the membrane patch under the glass pipette and achieve the whole-mitoplast configuration. After the break-in, fit the capacitance transients with the amplifier program's membrane test option to assess the membrane capacitance and its access resistance.Immediately after the break-in, replace the potassium chloride bath solution with a HEPES bath solution via perfusion. Apply an 850-millisecond ramp protocol with the amplifier program. Application of the voltage ramp protocol induces a large-amplitude proton current across the IMM of brown fat without the addition of exogenous fatty acids, the required activators of UCPs.After perfusion by either UCP1 inhibitor guanosine diphosphate or 10-millimolar methyl beta cyclodextrin for the endogenous membrane fatty acid extraction, the residual current is used to determine the amplitude of the UCP1 currents, which disappears completely in the IMM of UCP1-deficient mice. Unlike brown fat, the IMM of non-adipose tissues, such as skeletal muscle and heart, does not develop a measurable proton current immediately after the break-in. To induce a measurable proton current through AAC, it is essential to apply the HEPES bath solution containing one-to two-micromolar of exogenous fatty acids.To confirm that the measured proton current is carried by AAC, it is important to apply specific inhibitors, carboxyatractyloside, which almost completely inhibit proton current shown in the IMM of AAC1-deficient mice. A constant but never complete inhibition of proton leak is achieved only when ADP is present on both sides of the membrane to generate an active nucleotide exchange via AAC. The quality of the mitoplast preparation will influence the success rate in achieving the whole-mitoplast configuration.It is essential to optimize it. Once the molecular mechanism of mitochondrial thermogenesis is dissected with the patch clamp technique, measurement of mitochondrial oxygen consumption will demonstrate the importance of proton current and thermogenesis in intact mitochondria. This technique opens the way for researchers to explore all kinds of conductances, ions, and metabolites important for the functioning of mitochondria.

the-use-patch-clamp-technique-to-study-thermogenic-capacity

Research

JoVE Journal

Biology

3.5K visualizações.  University of California Los Angeles. Este protocolo descreve como a técnica de grampo de remendo pode ser usada para estudar a capacidade termogênica das mitocôndrias medindo diretamente o vazamento de prótons mitocondriais através da membrana mitocondrial interna. A medição direta da corrente de próton através da membrana mitocondrial interna ajuda a identificar e caracterizar precisamente os mecanismos moleculares responsáveis pela termogênese mitocondrial. Esta técnica permite não apenas medir a corrente de prót...