The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore

Abstract

A sudden increase in permeability of the inner mitochondrial membrane, the so-called mitochondrial permeability transition, is a common feature of apoptosis and is mediated by the mitochondrial permeability transition pore (mtPTP). It is thought that the mtPTP is a protein complex formed by the voltage-dependent anion channel, members of the pro-and anti-apoptotic BAX-BCL2 protein family, cyclophilin D, and the adenine nucleotide (ADP/ATP) translocators (ANTs) 1,2 . The latter exchange mitochondrial ATP for cytosolic ADP and have been implicated in cell death. To investigate the role of the ANTs in the mtPTP, we genetically inactivated the two isoforms of ANT [3][4][5] in mouse liver and analysed mtPTP activation in isolated mitochondria and the induction of cell death in hepatocytes. Mitochondria lacking ANT could still be induced to undergo permeability transition, resulting in release of cytochrome c. However, more Ca 2+ than usual was required to activate the mtPTP, and the pore could no longer be regulated by ANT ligands. Moreover, hepatocytes without ANT remained competent to respond to various initiators of cell death. Therefore, ANTs are non-essential structural components of the mtPTP, although they do contribute to its regulation.To investigate the role of ANTs in the mtPTP, we inactivated both the heart-muscle (Ant1) and the systemic (Ant2) ANT isoform genes in the mouse liver. Humans have three ANT genes 6,7 , whereas results of complementary DNA library screening 5 and northern and western analyses 3 have suggested that mouse has only two Ant genes. To verify this, we screened the Celera and Ensembl mouse genome assemblies, as well as the respective EST 1a). Targeted embryonic stem cells (Fig. 1b) were used to introduce the conditional floxed allele into the mouse germ line. Liver-specific inactivation of the Ant2 fl allele was achieved by breeding Ant2 fl mice with an Alb-Cre line of transgenic mice that expresses CRE under control of the liver-specific albumin promoter, which results in the excision of exons 3 and 4 of Ant2 (Fig. 1a) Endogenous respiration rates of ANT1/ANT2-deficient mitochondria were almost twice that of control mitochondria (34.58 ± 1.6 versus 18.12 ± 1.1 nmol O min -1 per mg protein) (Fig. 2b) and the mitochondrial membrane potential (ΔP = ΔΨ + ΔpH) of the ANT-deficient mitochondria was higher than that of controls (191.7 ± 4.9 versus 172.9 ± 3.5 mV). Analysis of the specific activity of OXPHOS enzyme complexes in ANT-deficient mitochondria revealed that complex IV (cytochrome c oxidase, COX) was increased more than twofold compared with controls (P < 0.01) (Fig. 2c). This was confirmed by western blot analysis, which revealed that the mitochondrial COX subunit I (COI) and cytochrome c proteins were more abundant in the ANT-deficient mitochondria (Fig. 2d). Hence, the increased respiration rate is likely to be the result of the specific upregulation in COX activity, suggesting that COX activity may modulate respiration rate. Because ΔP is the product of proton pumpin...

Mitochondria and aging: A role for the mitochondrial transition pore?

“…Indeed, cardiolipin interacts with and plays a key role for the transport of adenine nucleotides by ANT (Hoffmann, Stöckl, Schlame, Beyer, & Klingenberg, 1994), which has long been considered as a structural component of the mPTP. Although deletion experiments have challenged this hypothesis (Kokoszka et al., 2004), additional data demonstrate a role of ANT in facilitating mPTP opening (Halestrap & Richardson, 2015). The oxidation of cardiolipine, which is tightly bound to ANT, could modify its conformation and facilitate mPTP opening.…”

“…Many proteins were thought to form the core of the pore across the mitochondrial membrane but they have been successively ruled out by genetic modulation. This is the case for the voltage‐dependent anion channel (VDAC) and the translocator protein (TSPO) in the outer membrane (Baines, Kaiser, Sheiko, Craigen, & Molkentin, 2007; Kokoszka et al., 2004). Recent data propose a role for ATP synthase as the major component of a multiproteic complex (Bernardi, Rasola, Forte, & Lippe, 2015).…”

Multiple Pathways to the Same End: Mechanisms of Myonuclear Apoptosis in Sarcopenia of Aging

“…This finding underlines the major role postulated for CyPD in the formation of the mPTP, and suggests a higher susceptibility to mPTP opening at an older age. Indeed, it was demonstrated that mPTP formation and apoptotic cell death could still occur in the absence of either ANT [57] or VDAC [58]. In contrast, overexpression of CyPD induced mitochondrial swelling and spontaneous cardiac apoptosis, whereas mice lacking cardiac CyPD were protected from ischemia/reperfusion-induced cell death [59].…”

Mitochondria as therapeutic targets for cancer chemotherapy

“…Moreover, ANT channel formation is enhanced by Bax (Marzo et al, 1998a), and inhibited by Bcl-2 . However, mouse knockout studies led to the conclusion that ANT would not (always) be required for apoptotic MOMP (Kokoszka et al, 2004). Recent evidence suggests that some ANT isoforms (ANT1, ANT3) are apoptogenic while others are not (ANT2) (Schubert and Grimm, 2004;Zamora et al, 2004).…”