Name: The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria (Video) | JoVE Journal
Uploaded: 2021-05-03T15:00:00.000+00:00
Duration: 11 min 5 s
Description: Learn the patch-clamp technique to study mitochondrial thermogenesis by measuring proton leak across the inner membrane. This method enables precise characterization of molecular mechanisms governing energy metabolism and heat production.

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5 minutes

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 the lipid phase before harvesting the mitochondria. To avoid confusion, the two mitochondrial isolation procedures (one from the brown fat and the other from the heart) are detailed below.
1. Mitochondrial isolation from mouse interscapular brown fat (modified from Bertholet et al. 2020)15
<ol>
	<li>Euthanize C57BL/6 male mouse using CO2 asphyxiation and subsequent cervical dislocation, as recommended by the American Veterinary Medical Association Panel and the IACUC Committee.</li>
	<li>After positioning the mouse with its belly facing the table, spray alcohol to clean and wet the hair (modified from Mann et al., 2014)16.</li>
	<li>Make an incision of 2 cm in the upper back after grasping the skin with tweezers.</li>
	<li>Extract the brown interscapular fat of the mouse that corresponds to a two-lobed organ with a butterfly shape16.</li>
	<li>Transfer the brown fat to a 35 mm Petri dish filled with 5 mL of cold isolation buffer (Table 1) previously placed on ice.</li>
	<li>Clean the brown fat from the white fat under a binocular.</li>
	<li>Transfer the brown fat to a 10 mL beaker with 5 mL of cold isolation buffer (Table 1) to chop it up into thin pieces. Transfer to the ice-chilled 10 mL glass homogenizer (plastic material polytetrafluoroethylene (PTFE) pestle).</li>
	<li>Use an overhead stirrer to homogenize the pre-cut tissue on ice with six gentle strokes at a controlled speed of 275 rotations/min.</li>
	<li>Centrifuge the homogenate at 8,500 x g for 10 min at 4 &#176;C in a 15 mL ice-cold conical tube. Discard the supernatant containing the lipid phase.</li>
	<li>Resuspend the pellet in 5 mL of ice-cold isolation buffer (Table 1) and homogenize, a second time, the suspension on ice with six slow strokes at a speed of 275 rotation/min.</li>
	<li>Transfer the homogenate in a 15 mL ice-cold conical tube and centrifuge it at 700 x g for 10 min at 4 &#176;C to pellet all nuclei and unbroken cells.</li>
	<li>Collect the supernatant in a fresh 15 mL tube and place it on ice.</li>
	<li>Centrifuge the supernatant at 8,500 x g for 10 min at 4 &#176;C to obtain a pellet containing mitochondria.</li>
	<li>Resuspend pellet containing mitochondria in 3.8 mL of ice-cold hypertonic-mannitol buffer (Table 2) and incubate the mitochondrial suspension on ice for 10-15 min.</li>
</ol>
2. Mitochondrial isolation from the mouse heart (modified from Garg et al. 2019)17
<ol>
	<li>Euthanize C57BL/6 male mouse using CO2 asphyxiation and subsequent cervical dislocation, as recommended by the American Veterinary Medical Association Panel and the IACUC Committee.</li>
	<li>After positioning the mouse on its back, spray alcohol to clean and wet the hair. Then, make a 2 cm incision on the thorax after grasping the skin with tweezers.</li>
	<li>Dissect the heart from the animal&#39;s chest and rinse it to remove all the blood in a 10 mL beaker with 5 mL of the cold isolation solution (Table 1).</li>
	<li>Once the heart has been cleared of traces of blood, transfer it to another 10 mL beaker containing 5 mL of cold isolation buffer (Table 1) to chop it up into thin pieces. Then, transfer to an ice-chilled 10 mL glass homogenizer (PTFE pestle).</li>
	<li>Use an overhead stirrer to homogenize the pre-cut tissue on ice with six gentle strokes at a controlled speed of 275 rotations/min.</li>
	<li>Transfer the homogenate to a 15 mL ice-cold conical tube and centrifuge it at 700 x g for 10 min at 4 &#176;C to pellet nuclei and unbroken cells.</li>
	<li>Collect the supernatant in a fresh 15 mL tube and place it on ice.</li>
	<li>Centrifuge the supernatant at 8,500 x g for 10 min at 4 &#176;C to obtain a pellet containing mitochondria.</li>
	<li>Resuspend the mitochondrial pellet in 3.8 mL of ice-cold hypertonic-mannitol buffer (Table 2) and incubate the mitochondrial suspension on ice for 10-15 min.</li>
</ol>
3. Preparation of mitoplasts with a French Press for mechanical rupture of the OMM.
NOTE: The French press procedure allows the IMM to be released from the OMM with its integrity preserved, including the matrix and crista (Figure 2)18. Mitochondria are pre-incubated in a hypertonic-mannitol buffer (Table 2) and subjected to a lower pressure during the French press procedure to avoid any drastic stretching of the IMM when the OMM is ruptured.
<ol>
	<li>Fill the mitochondrial-hypertonic-mannitol suspension into a refrigerated mini pressure cell (piston diameter 3/8&#34;) of the French press (Figure 3A).</li>
	<li>Select the Medium mode of the French Press and compress the suspension through the mini pressure cell at 110 on the dial of the French Press for the brown fat mitochondria and at 140 for the heart mitochondria (~2,000 psi).&#160; Ensure that the suspension comes out of the mini pressure cell at a rate of about 1 drop/s.</li>
	<li>Collect the drops in a 15 mL ice-chilled conical tube.</li>
	<li>Centrifuge the suspension at 10,500 x g for 10 min at 4 &#730;C.</li>
	<li>Resuspend the mitoplasts pellet in 0.5-2 mL of ice-cold Hypertonic-KCl buffer (Table 3) and store the suspension on ice. 
	NOTE: Brown fat and heart mitoplasts are ready for patch-clamp recordings and should remain usable for about 3-6 h.</li>
</ol>
4. Electrophysiological recordings of H+ leak through UCP1 and AAC5,7,15
NOTE: Use the following electrophysiological setup (Figure 3B): inverted microscope with differential interference contrast (DIC), 60x water immersion objective, vibration isolation table and a Faraday cage, a standard amplifier supporting low-noise recordings, a standard digitizer used for electrophysiological setup, pClamp 10, a micromanipulator, bath reference electrode (3 M KCl-agar salt bridge inserted within a microelectrode holder containing a silver/silver chloride pellet molded into the holder body (described in Liu et al. 2021)19, perfusion chamber with a 0.13 mm glass coverslip bottom, connected to a gravity-fed perfusion system.
<ol>
	<li>Pull the borosilicate glass filaments on the day of recording using a micropipette puller. Set a program on the puller used for generating pipettes with a high degree of reproducibility20. 
	NOTE: This program design requires several tries to obtain pipettes optimized for the IMM patch-clamp. A standard pipette has fine tips with a progressive conic shape.</li>
	<li>Insert one glass filament within the puller and pull to obtain almost two identical patch pipettes from one borosilicate filament.</li>
	<li>Adjust the program when pipettes become inconsistent between pulling cycles due to aging of the heating box filament of the puller.</li>
	<li>Position the pipette inside the pipette polisher and place the tip near the filament under 100x magnification to fire-polish it.</li>
	<li>Press the foot pedal several times to heat the filament without clogging or damaging the tip curve.</li>
	<li>Polish until pipettes with a resistance between 25 and 35 M&#937; are obtained when filled with TMA-based pipette solution (TMA for tetramethylammonium hydroxide, Table 4).</li>
	<li>Pre-incubate coverslips (5 mm diameter, 0.1 mm thickness) with 0.1% gelatin to reduce the mitoplast adhesion and rinse them with the KCl bath solution (Table 5) before depositing the mitoplast suspension.</li>
	<li>Prepare a raw dilution by mixing ~35 &#181;L of the concentrated mitoplast suspension with 500 &#181;L of the KCl bath solution (Table 5) and place it on coverslips previously placed in a well of a 4-well plate.</li>
	<li>Incubate on ice for 15 to 20 min for mitoplasts to sediment on the coverslip.</li>
	<li>Fill the bath chamber completely with ~50 &#181;L of the KCl bath solution (Table 5).</li>
	<li>Transfer a coverslip with mitoplasts within the chamber using thin microdissection tweezers with a bent tip.</li>
	<li>Arrange the coverslip at the bottom of the chamber. Do not perfuse the chamber to keep the mitoplasts stable on the coverslip.</li>
	<li>Choose an individual non-adhesive mitoplast in the shape of 8 by scanning the coverslip under the microscope with a 60x objective.</li>
	<li>Load the pipette with the pipette solution (~50 &#181;L) and place it in the pipette holder.</li>
	<li>Bring the pipette into the bath solution with a micromanipulator and approach it just above the selected mitoplast to get close to the IMM. The amplifier program gives the resistance of the pipette once it is in the bath solution. Hold the membrane potential at 0 mV and apply 10 mV pulses using the membrane test command in the amplifier program.</li>
	<li>Apply slight negative pressure to quickly create a gigaseal with the IMM (Figure 2B).</li>
	<li>Rise the pipette with the mitoplast attached to keep them away from the coverslip to avoid the seal breakage due to the pipette drift during the experiment.</li>
	<li>Compensate the stray capacitance transients with the &#34;membrane test&#34; command in the amplifier program before testing the whole-mitoplast configuration to obtain a correct capacitance (Cm) measurement for the mitoplast membrane after the break-in.</li>
	<li>Apply short-duration (5-15 ms) voltage pulses (250-600 mV) with the amplifier program to rupture the membrane patch under the glass pipette and achieve the whole-mitoplast configuration (Figure 2C). Successful break-in is reflected by the reappearance of capacitance transients.</li>
	<li>After the break-in, fit the capacitance transients with the amplifier program&#39;s membrane test option to assess the membrane capacitance (reflecting the size of the mitoplast) and its access resistance Ra (reflecting the quality of the whole-mitoplast configuration). After the break-in, Ra should be between 40 and 80 M&#937;. Mitoplasts (2-6 &#181;m in size) used for patch-clamp experiments typically have membrane capacitances of 0.5-1.1 pF.</li>
	<li>Immediately after the break-in, replace the KCl bath solution (Table 5) with the HEPES bath solution (Table 6) by starting the perfusion.</li>
	<li>Apply an 850 ms ramp protocol designed with the amplifier program, from -160 mV to +100 mV with a 5 s interval, while holding the mitoplast at 0 mV. This protocol works for UCP16,7,15 and AAC5 studies (Figure 4 and Figure 5). 
	NOTE: It is recommended for UCP1 and AAC-dependent H+ current measurements to acquire all electrophysiological data at 10 kHz and to filter at 1 kHz using adequate software driving the amplifier and digitizer.</li>
</ol>

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<li>Cannon, B., Nedergaard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brown+adipose+tissue%3A+function+and+physiological+significance%5BTitle%5D)+AND+%22Physiological+Reviews%22%5BJournal%5D)">Brown adipose tissue: function and physiological significance.</a> Physiological Reviews. 84 (1), 277-359 (2004).</li>
<li>Nicholls, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+hunt+for+the+molecular+mechanism+of+brown+fat+thermogenesis%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">The hunt for the molecular mechanism of brown fat thermogenesis.</a> Biochimie. 134, 9-18 (2017).</li>
<li>Bertholet, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((H%28%2B%29+transport+is+an+integral+function+of+the+mitochondrial+ADP%2FATP+carrier%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">H(+) transport is an integral function of the mitochondrial ADP/ATP carrier.</a> Nature. 571 (7766), 515-520 (2019).</li>
<li>Fedorenko, A., Lishko, P. V., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+of+fatty-acid-dependent+UCP1+uncoupling+in+brown+fat+mitochondria%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Mechanism of fatty-acid-dependent UCP1 uncoupling in brown fat mitochondria.</a> Cell. 151 (2), 400-413 (2012).</li>
<li>Bertholet, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+patch+clamp+of+beige+adipocytes+reveals+UCP1-positive+and+UCP1-negative+cells+both+exhibiting+futile+creatine+cycling%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">Mitochondrial patch clamp of beige adipocytes reveals UCP1-positive and UCP1-negative cells both exhibiting futile creatine cycling.</a> Cell Metabolism. 25 (4), 811-822 (2017).</li>
<li>Kirichok, Y., Krapivinsky, G., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mitochondrial+calcium+uniporter+is+a+highly+selective+ion+channel%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The mitochondrial calcium uniporter is a highly selective ion channel.</a> Nature. 427 (6972), 360-364 (2004).</li>
<li>Fieni, F., Lee, S. B., Jan, Y. N., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activity+of+the+mitochondrial+calcium+uniporter+varies+greatly+between+tissues%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Activity of the mitochondrial calcium uniporter varies greatly between tissues.</a> Nature Communications. 3, 1317(2012).</li>
<li>Chaudhuri, D., Sancak, Y., Mootha, V. K., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MCU+encodes+the+pore+conducting+mitochondrial+calcium+currents%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">MCU encodes the pore conducting mitochondrial calcium currents.</a> eLife. 2, 00704(2013).</li>
<li>Vais, H., Payne, R., Paudel, U., Li, C., Foskett, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coupled+transmembrane+mechanisms+control+MCU-mediated+mitochondrial+Ca%282%2B%29+uptake%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Coupled transmembrane mechanisms control MCU-mediated mitochondrial Ca(2+) uptake.</a> Proceedings of the National Academy of Sciences of the United States of America. 117 (35), 21731-21739 (2020).</li>
<li>Vais, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EMRE+is+a+matrix+Ca%282%2B%29+sensor+that+governs+gatekeeping+of+the+mitochondrial+Ca%282%2B%29+uniporter%5BTitle%5D)+AND+%22Cell+Reports%22%5BJournal%5D)">EMRE is a matrix Ca(2+) sensor that governs gatekeeping of the mitochondrial Ca(2+) uniporter.</a> Cell Reports. 14 (3), 403-410 (2016).</li>
<li>Vais, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MCUR1%2C+CCDC90A%2C+is+a+regulator+of+the+mitochondrial+calcium+uniporter%5BTitle%5D)+AND+%22Cell+Metabolism%22%5BJournal%5D)">MCUR1, CCDC90A, is a regulator of the mitochondrial calcium uniporter.</a> Cell Metabolism. 22 (4), 533-535 (2015).</li>
<li>Kamer, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MICU1+imparts+the+mitochondrial+uniporter+with+the+ability+to+discriminate+between+Ca%282%2B%29+and+Mn%282%2B%29%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">MICU1 imparts the mitochondrial uniporter with the ability to discriminate between Ca(2+) and Mn(2+).</a> Proceedings of the National Academy of Sciences of the United States of America. 115 (34), 7960-7969 (2018).</li>
<li>Bertholet, A. M., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patch-clamp+analysis+of+the+mitochondrial+H%28%2B%29+leak+in+brown+and+beige+fat%5BTitle%5D)+AND+%22Frontiers+in+Physiology%22%5BJournal%5D)">Patch-clamp analysis of the mitochondrial H(+) leak in brown and beige fat.</a> Frontiers in Physiology. 11, 326(2020).</li>
<li>Mann, A., Thompson, A., Robbins, N., Blomkalns, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization%2C+identification%2C+and+excision+of+murine+adipose+depots%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Localization, identification, and excision of murine adipose depots.</a> Journal of Visualized Experiments: JoVE. (94), e52174(2014).</li>
<li>Garg, V., Kirichok, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patch-clamp+analysis+of+the+mitochondrial+calcium+uniporter%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Patch-clamp analysis of the mitochondrial calcium uniporter.</a> Methods in Molecular Biology. 1925, 75-86 (2019).</li>
<li>Decker, G. L., Greenawalt, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructural+and+biochemical+studies+of+mitoplasts+and+outer+membranes+derived+from+French-pressed+mitochondria.+Advances+in+mitochondrial+subfractionation%5BTitle%5D)+AND+%22Journal+of+Ultrastructure+Research%22%5BJournal%5D)">Ultrastructural and biochemical studies of mitoplasts and outer membranes derived from French-pressed mitochondria. Advances in mitochondrial subfractionation.</a> Journal of Ultrastructure Research. 59 (1), 44-56 (1977).</li>
<li>Liu, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recording+electrical+currents+across+the+plasma+membrane+of+mammalian+sperm+cells%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%3A+JoVE%22%5BJournal%5D)">Recording electrical currents across the plasma membrane of mammalian sperm cells.</a> Journal of Visualized Experiments: JoVE. (168), (2021).</li>
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</ol>

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><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&amp;nbsp; 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>&lt;strong&gt;Chemicals&lt;/strong&gt;</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>&amp;nbsp;Sigma Aldrich</td><td>M4125</td><td></td></tr><tr><td>EGTA</td><td>&amp;nbsp;Sigma Aldrich</td><td>3777</td><td></td></tr><tr><td>HEPES</td><td>&amp;nbsp;Sigma Aldrich</td><td>H7523</td><td></td></tr><tr><td>KCl</td><td>&amp;nbsp;Sigma Aldrich</td><td>60128</td><td></td></tr><tr><td>MgCl2</td><td>&amp;nbsp;Sigma Aldrich</td><td>63068</td><td></td></tr><tr><td>sucrose</td><td>&amp;nbsp;Sigma Aldrich</td><td>S7903</td><td></td></tr><tr><td>TMA</td><td>&amp;nbsp;Sigma Aldrich</td><td>331635</td><td></td></tr><tr><td>TrisBase</td><td>&amp;nbsp;Sigma Aldrich</td><td>T1503</td><td></td></tr><tr><td>TrisCl</td><td>&amp;nbsp;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

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...

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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Research

JoVE Journal

Biology

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of  F1FO ATPase complex and its modulation using the technique of patch clamp recording.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

One-channel Cell-attached Patch-clamp Recording

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

This method exploits the contribution of the mitochondrial permeability transition pore to low-conductance proton leak to determine the voltage threshold for pore opening in neonatal fragile X syndrome mice with increased cardiomyocyte mitochondrial coenzyme Q content compared to wildtype control.

Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess

Mice and rats are surgically implanted with remote temperature transponders and then habituated to the testing environment and procedure. Changes in muscle temperature are measured in response to pharmacological or contextual stimuli in the home cage or during prescribed physical activity (i.e., treadmill walking at a constant speed).

Measuring Skeletal Muscle Thermogenesis in Mice and Rats

Here, we present a protocol to quantify the physiological significance of the impact of brown adipose tissue (BAT) activity on human metabolism. This is achieved by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach can help develop a pharmacological target for BAT thermogenesis in humans.

Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans

This protocol details a method for directly measuring ion channel activity on intracellular vesicles using a manual endolysosomal patch-clamp system. We illustrate the method that involves enlarging endolysosomes and manually isolating these vesicles. This approach ensures that researchers can accurately replicate and apply the procedure.

Patch-Clamp Techniques for Single Endolysosomal Vesicle Analysis

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Learn the patch-clamp technique to study mitochondrial thermogenesis by measuring proton leak across the inner membrane. This method enables precise characterization of molecular mechanisms governing energy metabolism and heat production.

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

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