Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase

Abstract

F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.

Alisporivir Treatment Alleviates Mitochondrial Dysfunction in the Skeletal Muscles of C57BL/6NCrl Mice with High-Fat Diet/Streptozotocin-Induced Diabetes Mellitus

“…The excessive accumulation of Ca 2+ in the mitochondrial matrix and the overproduction of reactive oxygen species are the main triggers of the MPT pore opening in mitochondria [9]. The adenine nucleotide translocator and the ATP synthase are currently considered to be the main proteins involved in the formation of the pore channel and are possibly in alliance with the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane [10][11][12][13]. Cyclophilin D is a master regulator of the MPT pore, and the inhibitors of this protein cyclosporin A and alisporivir suppress the pore opening at submicromolar concentrations [12][13][14].…”

Modulation and Pharmacology of the Mitochondrial Permeability Transition: A Journey from F-ATP Synthase to ANT

“…In wild-type cells and mitoplasts, PTP opening could be efficiently inhibited by CsA, but was completely refractory to BKA, suggesting that the ANT pore (also referred to as A-PTP) did not emerge in the first place, while the F-ATP synthase pore (also referred to as F-PTP) appears to predominate. The lack of subunit b or OSCP, which generates vestigial F-ATP synthases [127] and likely prevents channel formation by the enzyme, unmasks the A-PTP, as confirmed by the efficacy of BKA in blocking channel activity in situ and the PT in living cells [130]. The ablation of subunit g, which also caused the loss of subunit e, completely prevented PTP opening unless ATR was added and forced A-PTP activation.…”

“…These findings pointed out that in the absence of a fully assembled F-ATP synthase, the PT could be mediated by ANT forming a distinct permeability pathway. In support of this hypothesis, Carrer et al provided further insights by analyzing the electrophysiological properties of mitoplasts isolated from HAP1 cells lacking subunit b or OSCP and from HeLa cells genetically ablated for subunit g [130]. In wild-type cells and mitoplasts, PTP opening could be efficiently inhibited by CsA, but was completely refractory to BKA, suggesting that the ANT pore (also referred to as A-PTP) did not emerge in the first place, while the F-ATP synthase pore (also referred to as F-PTP) appears to predominate.…”

“…The ablation of subunit g, which also caused the loss of subunit e, completely prevented PTP opening unless ATR was added and forced A-PTP activation. Different from mitoplasts devoid of subunit c, which showed channels sensitive to both BKA and CsA [129], mitoplasts with a defective F-ATP synthase peripheral stalk (i.e., absence of OSCP or b subunits) became sensitive to BKA but were refractory to CsA [130]. This puzzling observation could help to shed light on a potential structural relationship between ANT and the F-ATP synthase, which were shown to physically interact in the so-called "ATP synthasome" and respond in a still undefined manner to CyPD [131,132].…”

Mitochondrial Permeability Transition Causes Mitochondrial Reactive Oxygen Species- and Caspase 3-Dependent Atrophy of Single Adult Mouse Skeletal Muscle Fibers

“…Although research continues, current evidence suggests that the formation of the mPTP involves a Ca 2+ -dependent conformational change of the F o F 1 -ATP synthase to form the channel [32]. Furthermore, there is also evidence in patch-clamped mitochondrial membranes that mPTP activity requires the presence of at least one of the isoforms of the adenine nucleotide translocase (also known as the ADP/ATP carrier) [33], and recent work suggests the ANT may contribute to channel formation in the absence of an assembled F-ATP synthase [34]. A prolonged opening of the mPTP results in a collapse of the mitochondrial membrane potential, and can cause rupture of the outer mitochondrial membrane, leading to the release of both mROS and mitochondrial proteins, such as apoptosis inducing factor (AIF), Endonuclease G (EndoG), and cytochrome c [35].…”