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.
