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

Summary

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.

Abstract

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in health and disease. The mPTP mediates leakage of protons across the IMM during low-conductance opening and is specifically inhibited by cyclosporine A (CsA). Coenzyme Q (CoQ) is a regulator of the mPTP, and tissue-specific differences have been found in CoQ content and open probability of the mPTP in forebrain and heart mitochondria in a newborn mouse model of fragile X syndrome (FXS, Fmr1 knockout). We developed a technique to determine the voltage threshold for mPTP opening in this mutant strain, exploiting the role of the mPTP as a proton leak channel.

To do so, oxygen consumption and membrane potential (ΔΨ) were simultaneously measured in isolated mitochondria using polarography and a tetraphenylphosphonium (TPP⁺) ion-selective electrode during leak respiration. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP were precisely defined in the context of CoQ excess. This novel technique will permit future investigation for enhancing the understanding of physiological and pathological regulation of low-conductance opening of the mPTP.

Introduction

The mPTP mediates the permeability transition (PT), whereby the IMM becomes abruptly permeable to small molecules and solutes^1,2. This striking phenomenon is a distinct departure from the characteristic impermeability of the IMM, which is fundamental for establishing the electrochemical gradient necessary for oxidative phosphorylation³. PT, unlike other mitochondrial transport mechanisms, is a high-conductance, nonspecific, and nonselective process, allowing the passage of a range of molecules up to 1.5 kDa^4,5. The mPTP is a voltage-gated channel within the IMM whose opening alters ΔΨ, ATP production, calcium homeostasis, reactive oxygen species (ROS) production, and cell viability⁴.

At the pathologic extreme, uncontrolled and prolonged high-conductance opening of mPTP leads to the collapse of the electrochemical gradient, matrix swelling, depletion of matrix pyridine nucleotides, outer membrane rupture, release of intermembrane proteins (including cytochrome c), and ultimately, cell death^4,6. Such pathological mPTP opening has been implicated in cardiac ischemia-reperfusion injury, heart failure, traumatic brain injury, various neurodegenerative diseases, and diabetes^1,7. However, low-conductance mPTP opening is physiological in nature and, in contrast to high-conductance opening, does not lead to profound depolarization or mitochondrial swelling⁴.

Low-conductance opening of the pore restricts permeability to ~300 Da, allows the passage of protons independent of ATP synthesis, and is a potential source of physiological proton leak⁵. Physiologic mPTP opening causes a controlled decline in ΔΨ, increases electron flux through the respiratory transport chain, and results in a short burst or flash of superoxide, contributing to ROS signaling⁸. Regulation of such transient mPTP opening is important for calcium homeostasis and normal cellular development and maturation^4,9,10,11. Transient pore opening in developing neurons, for example, triggers differentiation, while the closure of the mPTP induces maturation in immature cardiomyocytes^4,5.

Although the functional significance of the mPTP in health and disease is well established, its precise molecular identity remains debated. Progress on the molecular structure and function of the mPTP has been comprehensively reviewed elsewhere¹². Briefly, currently, high- and low- conductance states of the mPTP have been hypothesized to be mediated by distinct entities¹². The leading candidates are the F1/F0 ATP synthase (ATP synthase) and adenine nucleotide transporter (ANT) for high- and low-conductance modes, respectively¹².

Despite the lack of consensus regarding the exact identity of the pore-forming component of the mPTP, certain key characteristics have been detailed. A well-established feature of the mPTP is that it is regulated by the electrochemical gradient such that depolarization of the IMM leads to pore opening¹³. Prior work has shown that the redox state of vicinal thiol groups alters the voltage gating of the mPTP, such that oxidation opens the pore at relatively higher ΔΨs, and thiol group reduction results in closed mPTP probability¹⁴. However, the identity of the proteinaceous voltage sensor is unknown.

Various small molecules that modulate the open probability of the pore have been identified. For example, the mPTP can be stimulated to open with calcium, inorganic phosphate, fatty acids, and ROS and can be inhibited by adenine nucleotides (particularly ADP), magnesium, protons, and CsA^5,12. The mechanisms of action of some of these regulators have been elucidated. Mitochondrial calcium triggers mPTP opening at least in part by binding to the β-subunit of the ATP synthase¹⁵. ROS can activate the mPTP by decreasing its affinity for ADP and enhancing its affinity for cyclophilin D (CypD), the best-studied proteinaceous mPTP activator¹⁶. The mechanism of activation of the mPTP by inorganic phosphate and fatty acids is less clear. As for endogenous inhibitors, ADP is thought to inhibit the mPTP by binding at the ANT or ATP synthase, while magnesium exerts its inhibitory effect by displacing calcium from its binding site^15,17,18,19.

Low pH inhibits mPTP opening by protonating histidine 112 of the regulatory oligomycin sensitivity-conferring protein (OSCP) subunit of the ATP synthase^12,20,21. The prototypical pharmacologic inhibitor of the mPTP, CsA, acts by binding CypD and preventing its association with OSCP^22,23. Previous work has also shown that a variety of CoQ analogs interact with the mPTP, inhibiting it or activating it²⁴. In recent work, we found evidence of a pathologically open mPTP, excessive proton leak, and inefficient oxidative phosphorylation due to a CoQ deficiency in forebrain mitochondria of newborn FXS mouse pups²⁵.

Closure of the pore with exogenous CoQ blocked the pathologic proton leak and induced morphologic maturity of dendritic spines²⁵. Interestingly, in the same animals, FXS cardiomyocytes had excessive CoQ levels and closed mPTP probability compared to wildtype controls²⁶. Although the cause of these tissue-specific differences in CoQ levels is unknown, the findings underscore the concept that endogenous CoQ is likely a key regulator of the mPTP. However, there is a major gap in our knowledge because the mechanism of CoQ-mediated inhibition of the mPTP remains unknown.

Regulation of the mPTP is a critical determinant of cell signaling and survival⁴. Thus, detecting mPTP opening within mitochondria is key when considering specific pathophysiological mechanisms. Typically, the threshold for high-conductance pore opening is determined using calcium to trigger the permeability transition. Such calcium loading leads to the collapse of the membrane potential, rapid uncoupling of oxidative phosphorylation, and mitochondrial swelling^27,28. We sought to develop a method to detect low-conductance mPTP opening in situ, without inducing it per se.

The approach exploits the role of the mPTP as a proton leak channel. To do so, Clark-Type and TPP⁺ ion-selective electrodes were employed to simultaneously measure oxygen consumption and membrane potential, respectively, in isolated mitochondria during leak respiration²⁹. The threshold for mPTP opening was determined by the onset of CsA-mediated inhibition of proton leak at specific membrane potentials. Using this approach, differences in voltage gating of the mPTP in the context of CoQ excess were precisely defined.

Protocol

Institutional Animal Care and Use Committee of Columbia University Medical Center approval was obtained for all methods described. FXS (Fmr1 KO) (FVB.129P2-Pde6b⁺ Tyr^c-ch Fmr1^tm1Cgr/J) and control (FVB) (FVB.129P2-Pde6b⁺ Tyr^c-ch/AntJ) mice used as the model systems for this study were commercially acquired (see the Table of Materials). Five to eleven animals were used in each experimental group. Postnatal day 10 (P10) mice were used to model for a time point in human infancy.

1. Mitochondrial isolation from mouse heart

1. Prepare buffers for mitochondrial isolation and respiration experiments as described in Table 1. Store at 4 °C if made in advance.
   1. Prepare 15% density gradient medium: dilute commercial density gradient medium to 80% volume/volume (v/v) with density gradient diluent. Further dilute 80% density gradient medium 15% v/v by adding mitochondrial isolation buffer (MI)/bovine serum albumin (BSA).
2. Isolate mitochondria from fresh, not frozen tissue for these experiments and use on the day of preparation, typically within five hours²⁶. Carry out all isolation steps on ice.
   1. Decapitate the mouse and excise the heart. Immediately wash 1-2 times in a Petri dish with ice-cold MI/BSA. Cut off the atrium and mince the tissue.
   2. Transfer the minced heart tissue to a glass-glass homogenizer containing 1 mL of MI/BSA. Homogenize with a looser (A) pestle (10 strokes), then a tighter (B) pestle (10 strokes).
   3. Centrifuge the homogenate at 1,100 × g for 2 min at 4 °C to remove nuclear and cellular debris.
   4. Take the supernatant gently without touching any fluffy pellet, and carefully layer it on top of 700 μL of 15% density gradient medium in a centrifuge tube. Centrifuge at 18,500 × g for 15 min at 4 °C.
   5. Resuspend the pellet in 1 mL of sucrose buffer (SB)/BSA and centrifuged at 10,000 × g for 10 min at 4 °C.
   6. Completely remove and discard the supernatant. Resuspend the pellet in SB/BSA to a final volume of 55 μL.
   7. Quantify mitochondrial protein content using a standard assay such as the bicinchoninic acid (BCA) protein assay.

2. Mitochondrial oxygen (O₂) consumption and ΔΨ

1. Oxygen electrode assembly and calibration
   1. Set up and calibrate the oxygen electrode (see the Table of Materials) according to the manufacturer's instructions.
      1. Place a drop of 50% potassium chloride (KCl) electrolyte solution on top of the dome of the electrode disc.
      2. Place a small piece ~2 cm² cigarette paper spacer covered with a slightly larger piece of the provided polytetrafluoroethylene (PTFE) membrane over the electrolyte drop.
      3. Using the applicator tool provided, push the small electrode disc O-ring over the dome of the electrode.
         NOTE: Ensure that there are no air bubbles and that the membrane is smooth.
   2. Top up the reservoir well with the electrolyte solution.
   3. Place the larger O-ring in the recess around the electrode disc well.
   4. Install the disc into the electrode chamber and connect it to the control unit.
   5. Add 2 mL of air saturated deionized water to the reaction chamber and the PTFE-coated magnet to the chamber.
   6. Connect the chamber to the rear of the control unit.
   7. Set the temperature to 37 °C and the stirring speed to 100.
   8. Allow 10 min for the system temperature to equilibrate before commencing calibration.
   9. Under the Calibration tab, select Liquid Phase Calibration to perform liquid phase calibration. Confirm that the temperature is set to 37 °C , the stirring speed is 100, and the pressure is set to atmospheric pressure (101.32 kPa).
   10. Press OK and wait for the signal to plateau.
   11. When a plateau is reached, press OK.
   12. Establish zero O₂ in the chamber by adding a small amount (~20 mg) of sodium dithionite.
   13. Press OK and wait for the signal to plateau.
   14. When a plateau is reached, press Save to accept the calibration.
2. TPP⁺-selective electrode assembly
   1. Fill the TPP⁺- selective electrode tip with 10 mM TPP solution using the provided syringe and flexible needle, taking care to avoid air bubbles.
   2. Assemble the TPP⁺-selective electrode apparatus, including the reference electrode and electrode holder, (see Table of Materials) according to the manufacturer's instructions.
   3. Briefly, loosen the electrode holder cap and insert the internal reference electrode into the TPP⁺ tip. Tighten the cap to secure the tip. Connect the provided cable to the electrode holder and the auxiliary port of the control box.
   4. Insert the TPP⁺-selective electrode and reference electrodes into the adapted plunger assembly for ion-selective electrodes. Connect the reference electrode to the reference port of the control box.
   5. Insert the TPP⁺-selective and reference electrodes into the adapted plunger assembly for ion-selective electrodes.
3. Reaction chamber preparation
   1. Add the reaction mixture to the reaction chamber: 10 mM succinate (complex II substrate), 5 μM rotenone (complex I inhibitor), 80 ng mL⁻¹ nigericin (to collapse pH gradient across IMM), 2.5 μg mL^-1 oligomycin (to induce state 4 respiration), and respiration buffer (RB)/BSA reaction buffer to a final volume of 1 mL. Take care to avoid introducing air bubbles into the chamber.
   2. Close the chamber with the adapted plunger assembly with the TPP⁺-selective and the reference electrode in place. Select GO to start recording. Once the chamber is closed, introduce additional reagents directly into the reaction solution using separate microsyringes modified with plastic tubing to adjust the needle length.
4. TPP⁺ calibration
   1. Once a stable voltage signal is obtained, calibrate the TPP⁺-selective electrode by adding 1 μM increments of a 0.1 mM TPP solution to a final concentration of 3 μM. Observe that there is a logarithmic decline in the TPP⁺ voltage signal with each addition.
      NOTE: Calibrate the TPP⁺-selective electrode at the start of each experiment.
5. Data acquisition
   1. Allow the O₂ and TPP⁺ traces to stabilize, and add 100 μg of freshly prepared cardiomyocyte mitochondria to the reaction chamber to a final concentration of 0.1 mg mL^-1 via the reagent addition port in the plunger assembly. Observe the decrease in the O₂ levels in the chamber as mitochondria become energized and consume O₂ and the abrupt increase in the TPP⁺ voltage signal as the mitochondria generate a membrane potential and take up TPP⁺ from the solution.
   2. Add 2.5 μg mL^-1 oligomycin to induce state 4 respiration.
      NOTE: O₂ consumption rates will now represent the rate of proton leak respiration. Oligomycin can be added to the reaction chamber prior to TPP⁺ as an alternative.
6. Assessing the open probability of the mPTP
   1. Observe that ΔΨ declines over time during leak respiration. Once the desired ΔΨ is reached, add 1 μM CsA (mPTP inhibitor) to the reaction chamber to assess open probability of the mPTP at that specific ΔΨ.
   2. Measure the effect of CsA on O₂ consumption and ΔΨ before and after CsA addition
   3. Determine the voltage dependence of mPTP opening by varying the ΔΨ at which CsA is added in successive experiments.

Results

Typical O₂ consumption and ΔΨ curves generated in these experiments are shown (Figure 1A,B). The logarithmic decline in the voltage signal with TPP⁺ calibration is shown at the start of each experiment. The absence of this logarithmic pattern may suggest a problem with the TPP⁺ selective electrode. Mitochondria typically generate ΔΨ immediately upon addition to respiratory buffer. ΔΨ can be interpreted from changes in TPP⁺ voltage ...

Discussion

This paper describes a method to assess the open probability of the mPTP. Specifically, the voltage threshold for low-conductance mPTP opening was determined by assessing the effect of CsA inhibition on proton leak over a range of ΔΨs. Using this technique, we could identify differences in voltage gating of the mPTP between FXS mice and FVB controls consistent with their differences in tissue-specific CoQ content. Critical to the success of this methodology is that mitochondria are freshly isolated prior to use...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)	Fisher Scientific	15630080
Adapted plunger assembly for pH or ion-selective electrodes for use with OXYT1	PP systems	941039
BD Intramedic PE Tubing, PE 50, 0.023 in. 10 ft.	Fisher Scientific	14-170-11B	to modify the length of the hamilton synringe as needed
Bovine Serum Albumin (BSA). Fatty acid free	Sigma	A7030-10G
Dri-Ref Reference Electrode, 2 mm	World Precision Inst. LLC	DRIREF-2
Electrode Holder for KWIK-Tips	World Precision Inst. LLC	KWIK-2	ion selective electrode holder
Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid  (EGTA)	Sigma	324626
FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J	Jackson Laboratory, Bar Harbor, ME		FXS mice, Fmr1 KO
FVB.129P2-Pde6b+ Tyrc-ch/AntJ	Jackson Laboratory, Bar Harbor, ME		FVB mice
Hamilton 80366 Standard Syringes, 10 uL, Cemented-Needle, 6/pk	Cole-Parmer	EW-07938-30	microsyringe
Hamilton 80500 Standard Microliter Syringes, 50 uL, Cemented-Needle	Cole-Parmer	EW-07938-02	microsyringe
Hansatech Instruments Oxytherm+ System (Respiration) Complete	PP systems	OXYTHERM+R	oxygen electrode and software
Magnesium Chloride (MgCl2)	Sigma	1374248
Mannitol	Sigma	M9546-250G
P1,P5-diadenosine-5′ pentaphosphate pentasodium (AP5A)	Sigma	D4022-10MG
Percoll	Sigma	P1644	medium for density gradient separation
Potassium chloride (KCl)	Sigma	P3911
Potassium dihydrogen phosphate (KH2PO4)	Sigma	5.43841
Sucrose	Sigma	S0389
TPP+ Electrode Tips (3)	World Precision Inst. LLC	TIPTPP