This protocol provides a new way to investigate the low conductance opening of the mitochondrial permeability transition pore. The main advantage of this technique is that it provides an easy way to measure the open probability of the pore as a function of mitochondrial membrane potential. This technique can be readily applied to mitochondria isolated from any organ system and species.
Begin with the calibration of the oxygen electrode by placing a drop of 50%potassium chloride electrolyte solution on top of the dome of the electrode disc. Place a small piece of two square centimeter cigarette paper spacer covered with a slightly larger piece of polytetrafluoroethylene membrane over the electrolyte drop. Then use the applicator tool to push the small electrode disc O-ring over the dome of the electrode.
Next, top up the reservoir well with the electrolyte solution. Place the larger O-ring in the recess around the electrode disc well. Then install the disc into the electrode chamber, and connect it to the control unit.
Add two milliliters of air-saturated deionized water, and the polytetrafluoroethylene-coated magnet to the reaction chamber. When done, connect the chamber to the rear of the control unit. Set the temperature to 37 degrees Celsius, and the stirring speed to 100.
Allow the system temperature to equilibrate for 10 minutes before calibration. After ensuring the correct temperature, stirring speed, and pressure, under the Calibration tab, select the Liquid Phase Calibration option to perform liquid phase calibration. Then press OK, and wait for the signal to plateau.
Once the plateau is reached, press OK.Then add about 20 milligrams of sodium dithionite to establish zero oxygen in the chamber. Again, press OK, and wait for the signal to plateau before clicking the Save button to accept the calibration. To prepare the tetraphenylphosphonium, or TPP-selective electrode assembly, fill the TPP-selective electrode tip with a 10 millimolar TPP solution using a syringe and a flexible needle.
Avoid air bubbles while filling the electrode tip. Loosen the electrode holder cap to insert the internal reference electrode into the TPP tip. When done, assemble the TPP-selective electrode apparatus, including the reference electrode and electrode holder.
Tighten the cap to secure the tip. Then connect the cable to the auxiliary port of the control box, and the TPP electrode holder. Insert the TPP-selective and reference electrodes into the adapted plunger assembly for ion-selective electrodes.
Connect the reference electrode to the reference port of the control box. Next, to prepare the reaction chamber, add one milliliter of the reaction mixture to the reaction chamber without creating air bubbles. Close the chamber using the adapted plunger assembly with the TPP-selective and reference electrodes in place.
Once the chamber is closed, introduce additional reagents directly into the reaction chamber using separate micro-syringes modified with plastic tubing. When the setup is ready, select Go to start recording. Once a stable voltage signal is obtained, calibrate the TPP-selective electrode by adding one micromolar increments of a 0.1 millimolar TPP solution to achieve a final concentration of three micromolar.
Observe the logarithmic decline in the TPP voltage signal with each addition. After stabilizing oxygen and TPP traces, add 100 micrograms of freshly prepared cardiomyocyte mitochondria to the reaction chamber via the reagent addition port in the plunger assembly to a final concentration of 0.1 milligrams per milliliter. Observe the decrease in the oxygen levels in the chamber as the mitochondria become energized and consume oxygen.
Also, see the abrupt increase in the TPP voltage signal as the mitochondria generates a membrane potential and take up TPP from the solution. Then add 2.5 micrograms per milliliter oligomycin to induce State 4 respiration. To assess the open probability of the mitochondrial permeability transition pore, or mPTP, observe the decline in the membrane potential over the time during leak respiration.
Once the desired membrane potential is reached, add one micromolar cyclosporine A as an mPTP inhibitor to the reaction chamber to assess the open probability of the mPTP at that specific membrane potential. Then measure the effect of cyclosporine A on oxygen consumption and membrane potential before and after cyclosporine A addition. In the representative curves of simultaneous oxygen consumption and membrane potential, high membrane potential was set at zero, intermediate at five, and low at 10 millivolts relative to the two micromolar TPP calibration level.
The mitochondria at zero millivolts exhibited 100%mPTP closed probability, and at 10 millivolts exhibited 100%open probability. After oligomycin addition, a significant difference in oxygen consumption was not observed, suggesting that adenosine triphosphate, or ATP synthase, contributes minimally to State 2 respiration. Further, the oxygen consumption rate and membrane potential before and after cyclosporine A addition were compared.
A decrease in the oxygen consumption rate and an increase in stabilization of membrane potential indicated the closure of an open mPTP. When the mPTP is closed, there was no decrease in oxygen consumption rate, and the membrane potential continued to fall. The results demonstrated similar closed and open mPTP probabilities at high and low membrane potentials in the FVB control and Fmr1 knockout cardiac mitochondria.
At the intermediate membrane potential, the Fmr1 knockout cardiac mitochondria demonstrated increased closed mPTP probability compared to the FVB controls. No air bubbles must be introduced into the chamber, as this will lead to unstable oxygen consumption readings, and make interpretation difficult. Following this procedure, calcium loading capacity can be measured.
This method assesses the high conductance opening of the pore, and complements the results of our technique.
