Name: assessment of open probability of the mitochondrial permeability transition pore in the setting of coenzyme q excess
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This work is supported by the following grants: NIH/NIGMS T32GM008464 (K.K.G.), Columbia University Irving Medical Center Target of Opportunity Provost award to the Department of Anesthesiology (K.K.G.), Society of Pediatric Anesthesia Young Investigator Research Award (K.K.G.), and NIH/NINDS R01NS112706 (R.J.L.)

Institutional Animal Care and Use Committee of Columbia University Medical Center approval was obtained for all methods described. FXS (Fmr1 KO) (FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J) and control (FVB) (FVB.129P2-Pde6b+ Tyrc-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...

<ol>
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A., Hunter, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ca2+-induced+membrane+transition+in+mitochondria.+II.+Nature+of+the+Ca2++trigger+site.">The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.</a> Archives of Biochemistry and Biophysics. 195 (2), 460-467 (1979).</li><li>Halestrap, A. P., Connern, C. P., Griffiths, E. J., Kerr, P. 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J., Murphy, E., Liu, J. C., Palmeira, C. M., Moreno, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Mitochondrial Bioenergetics: Methods and ProtocolsMethods in Molecular Biology. , 187-196 (2018).</li><li>Carraro, M., Bernardi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+membrane+permeability+and+the+mitochondrial+permeability+transition.">Measurement of membrane permeability and the mitochondrial permeability transition.</a> Methods in Cell Biology. 155, 369-379 (2020).</li><li>Affourtit, C., Wong, H., Brand, M. D., Palmeira, C. M., Moreno, A. 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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 &#916;&#936;s. 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...

The mPTP mediates the permeability transition (PT), whereby the IMM becomes abruptly permeable to small molecules and solutes1,2. 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 kDa4,5. The mPTP is a voltage-gated channel within the IMM whose opening alters &#916;&#936;, 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 death4,6. Such pathological mPTP opening has been implicated in cardiac ischemia-reperfusion injury, heart failure, traumatic brain injury, various neurodegenerative diseases, and diabetes1,7. 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 &#916;&#936;, 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 maturation4,9,10,11. Transient pore opening in developing neurons, for example, triggers differentiation, while the closure of the mPTP induces maturation in immature cardiomyocytes4,5.
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 &#916;&#936;s, 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 CsA5,12. 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 &#946;-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 site15,17,18,19.
Low pH inhibits mPTP opening by protonating histidine 112 of the regulatory oligomycin sensitivity-conferring protein (OSCP) subunit of the ATP synthase12,20,21. The prototypical pharmacologic inhibitor of the mPTP, CsA, acts by binding CypD and preventing its association with OSCP22,23. 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 swelling27,28. 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.

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	<tbody>
		<tr>
			<td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td>
			<td>Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>Adapted plunger assembly for pH or ion-selective electrodes for use with OXYT1</td>
			<td>PP systems</td>
			<td>941039</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Intramedic PE Tubing, PE 50, 0.023 in. 10 ft.</td>
			<td>Fisher Scientific</td>
			<td>14-170-11B</td>
			<td>to modify the length of the hamilton synringe as needed</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA). Fatty acid free</td>
			<td>Sigma</td>
			<td>A7030-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dri-Ref Reference Electrode, 2 mm</td>
			<td>World Precision Inst. LLC</td>
			<td>DRIREF-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode Holder for KWIK-Tips</td>
			<td>World Precision Inst. LLC</td>
			<td>KWIK-2</td>
			<td>&#160;ion selective electrode holder</td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid&#160; (EGTA)</td>
			<td>Sigma</td>
			<td>324626</td>
			<td></td>
		</tr>
		<tr>
			<td>FVB.129P2-Pde6b+ Tyrc-ch Fmr1tm1Cgr/J</td>
			<td>Jackson Laboratory, Bar Harbor, ME</td>
			<td></td>
			<td>FXS mice, Fmr1 KO&#160;</td>
		</tr>
		<tr>
			<td>FVB.129P2-Pde6b+ Tyrc-ch/AntJ</td>
			<td>Jackson Laboratory, Bar Harbor, ME</td>
			<td></td>
			<td>FVB mice</td>
		</tr>
		<tr>
			<td>Hamilton 80366 Standard Syringes, 10 uL, Cemented-Needle, 6/pk</td>
			<td>Cole-Parmer</td>
			<td>EW-07938-30</td>
			<td>microsyringe</td>
		</tr>
		<tr>
			<td>Hamilton 80500 Standard Microliter Syringes, 50 uL, Cemented-Needle</td>
			<td>Cole-Parmer</td>
			<td>EW-07938-02</td>
			<td>microsyringe</td>
		</tr>
		<tr>
			<td>Hansatech Instruments Oxytherm+ System (Respiration) Complete</td>
			<td>PP systems</td>
			<td>OXYTHERM+R</td>
			<td>oxygen electrode and software</td>
		</tr>
		<tr>
			<td>Magnesium Chloride (MgCl2)</td>
			<td>Sigma</td>
			<td>1374248</td>
			<td></td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M9546-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>P1,P5-diadenosine-5&#8242; pentaphosphate pentasodium (AP5A)</td>
			<td>Sigma</td>
			<td>D4022-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>P1644</td>
			<td>medium for density gradient separation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate (KH2PO4)</td>
			<td>Sigma</td>
			<td>5.43841</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S0389</td>
			<td></td>
		</tr>
		<tr>
			<td>TPP+ Electrode Tips (3)</td>
			<td>World Precision Inst. LLC</td>
			<td>TIPTPP</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of open probability of the mitochondrial permeability transition pore in the setting of coenzyme q excess

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 (&#916;&#936;) 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.

Typical O2 consumption and &#916;&#936; 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 &#916;&#936; immediately upon addition to respiratory buffer. &#916;&#936; can be interpreted from chang...

Watch this Scientific Journal Video about Assessment of Open Probability of the Mitochondrial Permeability Transition Pore in the Setting of Coenzyme Q Excess at JoVE.com

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

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.

The mitochondrial permeability transition pore (mPTP) is a voltage-gated, nonselective, inner mitochondrial membrane (IMM) mega-channel important in ...

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