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Biology

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

Published: May 3rd, 2021

DOI:

10.3791/62618

1Department of Physiology, David Geffen School of Medicine, University of California Los Angeles

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 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.

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....

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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 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.......

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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.......

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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.

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

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