Quantification of active mitochondrial permeability transition pores using GNX-4975 inhibitor titrations provides insights into molecular identity

Andrew P Richardson,Andrew P Halestrap

doi:10.1016/j.bbabio.2016.04.367

Andrew P Richardson, Andrew P Halestrap

Open Access

https://doi.org/10.1016/j.bbabio.2016.04.367

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Abstract

Inhibition of the mitochondrial permeability transition pore (MPTP) by the novel inhibitor GNX-4975 was characterized. Titration of MPTP activity in de-energized rat liver mitochondria allowed determination of the number of GNX-4975-binding sites and their dissociation constant ( K i). Binding sites increased in number when MPTP opening was activated by increasing [Ca2+], phenylarsine oxide (PAO) or KSCN, and decreased when MPTP opening was inhibited with bongkrekic acid (BKA) or ADP. Values ranged between 9 and 50 pmol/mg of mitochondrial protein, but the K i remained unchanged at ∼1.8 nM when the inhibitor was added before Ca2+. However, when GNX-4975 was added after Ca2+ it was much less potent with a K i of ∼140 nM. These data imply that a protein conformational change is required to form the MPTP complex and generate the GNX-4975-binding site. Occupation of the latter with GNX-4975 prevents the Ca2+ binding that triggers pore opening. We also demonstrated that GNX-4975 stabilizes an interaction between the adenine nucleotide translocase (ANT), held in its ‘c’ conformation with carboxyatractyloside (CAT), and the phosphate carrier (PiC) bound to immobilized PAO. No components of the F1Fo-ATP synthase bound significantly to immobilized PAO. Our data are consistent with our previous proposal that the MPTP may form at an interface between the PiC and ANT (or other similar mitochondrial carrier proteins) when they adopt novel conformations induced by factors that sensitize the MPTP to [Ca2+]. We propose that GNX-4975 binds to this interface preventing a calcium-triggered event that opens the interface into a pore. * ANT, : adenine nucleotide translocase; BKA, : bongkrekic acid; CAT, : carboxyatractyloside; CsA, : cyclosporin A; CyP-D, : cyclophilin D; IMM, : inner mitochondrial membrane; ISB, : isolation buffer; MCF, : mitochondrial carrier family; MPTP, : mitochondrial permeability transition pore; NTA, : nitrilotriacetic acid; PAO, : phenylarsine oxide; PCB, : PAO column buffer; PEG, : polyethylene glycol; PiC, : phosphate carrier; SPG7, : spastic paraplegia 7

Highlights

The mitochondrial permeability transition pore (MPTP) is a large non-specific channel in the inner mitochondrial membrane (IMM) whose opening is triggered by high matrix [Ca2+ ] to which it can be sensitized by a variety of factors
cyclophilin D (CyP-D) is the site of action of two potent inhibitors of MPTP opening, cyclosporin A (CsA) and sanglifehrin A (SfA), both of which protect cells from death mediated by MPTP opening such as in ischaemia/reperfusion injury [3]
In order to determine an accurate concentration-dependence of MPTP inhibition by GNX-4975, we employed de-energized mitochondria in the presence of a Ca2+ ionophore (A23187), since this avoids any complications that might be caused by secondary effects of the drug on mitochondrial energization or calcium transport

Summary

Introduction

The mitochondrial permeability transition pore (MPTP) is a large non-specific channel in the inner mitochondrial membrane (IMM) whose opening is triggered by high matrix [Ca2+ ] to which it can be sensitized by a variety of factors. These include elevated [Pi], adenine nucleotide depletion, agents such as carboxyatractyloside (CAT) that stabilize the adenine nucleotide translocase (ANT) in its cytoplasmic facing (‘c’) conformation, mild chaotropic agents such as KSCN and oxidative stress or its chemical mimic phenylarsine oxide (PAO) [1]. The molecular composition of the IMM components of the MPTP, with which CyP-D interacts, remains controversial

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