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 vol....

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.......

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

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