*4.4. How the ATP Synthase/Hydrolase Is Involved in the Mitochondrial Permeability Transition Pore*

In recent years, the F1FO-ATPase has been implicated in the mPTP, a large pore in the mtIM, which permeabilizes the mtIM to ions and other solutes [178–180]. As the membrane becomes permeable, the massive water influx in the matrix results in mitochondrial swelling. For short full openings, the mPTP is reversible, but prolonged openings are irreversible and trigger the release of cyt. *c* and other pre-apoptotic factors which drive the cell to death [181]. The mPTP formation is stimulated by ROS and Ca2+ increase, by the binding of the mitochondrial protein cyclophilin D (CyPD) in mitochondria and inhibited by H<sup>+</sup> , Mg2+, adenine nucleotides, and cyclosporin A (CsA), which displaces CyPD and desensitizes the mPTP to Ca2+ [182]. The mPTP activity mainly appeared as a property of mammalian and yeast mitochondria, where it was intensively investigated. However recently the mPTP was also reported in mussel [183], in sea urchin oocytes [184], in the nematode *Caenorhabditis elegans* [185], in plathelminths [186] and insects [187,188], while it is still controversial in crustaceans [189,190]. Indeed, it is not a "vertebrate invention" to rule cell fate, as initially thought [191].

At present, the problem of the involvement of F1FO-ATPase in the mPTP and especially its structural participation in the mechanism of mPTP formation and opening is near to being solved [128]. This quite surprising task of the F1FO-ATPase, which has been supposed by different research groups, points out the versatility of this intriguing enzyme complex, which emerges as the playmaker of cell life and cell death. Two main F1FO-ATPase sites have been involved in the mPTP formation: the *c*-ring [179,180] or the monomer–monomer interface of the dimer [178].

The hydrolytic function of the F1FO-ATPase, namely ATP hydrolysis, can be sustained by metal divalent cations different from the natural cofactor Mg2+, such as Ca2+, which is unable to activate the enzyme complex in the opposite function of ATP synthesis [192–195]. Interestingly, Ca2+ rise in the mitochondrial matrix initiates a cascade of events which lead to cell death. So, in recent times Ca2+ binding to the F1FO-ATPase in replacement of Mg2+ , an event which can easily occur in the presence of relatively high Ca2+ concentrations in mitochondria, since the enzyme affinity for Ca2+ is lower than that for Mg2+ [193,196], has been associated with the mPTP opening [192,195,197–199].

Accordingly, the structure of mammalian F1FO-ATPase exposed to Ca2+ and revealed by cryo-electron microscopy, shows unusual states, not identified when Mg2+ acts as cofactor, which can be ascribed to mPTP opening. Indeed, in the new "bent-pull" model, the Sazanov lab highlights the role of *c*-ring and of some *sms* in mPTP formation. Accordingly, when Ca2+ replaces Mg2+ in the catalytic binding site, the cation insertion in the Ca2+-activated F1FO-ATPase, due to the higher steric hindrance, would promote conformational changes of F<sup>1</sup> domain, in turn transmitted by the peripheral stalk [198] to membrane subunits that form the mPTP [198]. Notably, when the Ca2+-activated F1FO-ATPase activity is decreased by various inhibitors, the mPTP formation is delayed or even prevented [196,197,200]. The *c*-ring contains two different phospholipids at the two opposite sides of its cavity. At the matrix side, phosphatidyl serine is anchored by ionic interactions to the positive charge of Arg-38 of *c* subunits, while at the intra*crista* side, Lys-71 of *e* subunit coordinates a lyso-phosphatidylserine. Since the phosphatidylserine double acyl-chain does not have enough space around the *c*-ring plug and is linked to the *c*-ring, it rotates together with the rotor. Conversely, the monoacyl chain of lysophosphatidylserine acts as "lubricated" lipid plug. The two lipids inside the *c*-ring are separated by the conserved *c*Val-16 [128]. The F1FO-ATPase distortion and tilt induced by Ca2+ trigger changes in the conformational states of the Ca2+-activated F1FO-ATPase that open the mPTP. Most likely, the signal propagation from F<sup>1</sup> to F<sup>O</sup> through the long helix of *b* subunit modifies the TTMHB assembly and changes the position of *e* subunits, which expel the lyso-phosphatidylserine from the central hole inside the *c*-ring and opens the channel at the positive mtIM side [181]. Consequently, the curved *crista* ridges and the F1FO-ATPase dimerization are lost [177,199,201]. According to Sazanov's hypothesis, the water molecules inside the *c*-ring destabilize the phosphatidylserine which pushes out the lipid plug and creates a pore through the *c*-ring, while the consequent conformational change detaches F<sup>1</sup> from FO. Thus, structural/conformational F1FO-ATPase changes are involved in the (ir)reversible mechanism of the mPTP, which opens and closes and rules cell fate. The most recent data strongly sustain the hypothesis that these changes mainly involve the *c*-ring, which emerges as the main character in mPTP formation (Figure 5).

However, this matter is still hot and some debate on this topic remains. The possibility that differently sized pores can be formed and coexist in the mtIM satisfactorily combines the conclusions drawn from different experimental approaches. Accordingly, the mtIM could be depolarized by any increase in conductance upon mitochondrial Ca2+ overload, due to transmembrane channels and/or transporters [202]. In this scenario, the Ca2+-activated F1FOATPase would contribute to the membrane depolarization by inducing the largest mPTP pore. It seems reasonable to think that smaller sub-conductance activities, which contribute to the mPTP can be ascribed to many other mitochondrial channels/transporters [203]. However, the mitochondrial F1FO-ATPase remains the most likely candidate as main high-conductance major channel or mPTP by definition, proven to be inhibited by CsA, but not by bongkrekate (BKA), which is known as inhibitor of the adenine nucleotide translocase [202,204].

Moreover, the adenine nucleotide translocase isoforms could form a secondary lowconductance mPTP inhibited by both CsA and BKA [205]. Notably, the mPTP activity can be enhanced by Ca2+ and pharmacologically modulated by selective inhibitors of F<sup>1</sup> [196] and F<sup>O</sup> domain [206–208]. Newly, purified and functionally active F1FO-ATPase monomers [209] and dimers [210] act as a voltage-gated ion channel endowed with mPTPlike properties.

Δ **Figure 5.** Model of mitochondrial permeability transition pore (mPTP) formation from the Ca2+-activated F1FO-ATPase. On the left Ca2+ bound to the catalytic sites activates the enzyme by triggering the structural change which opens the mPTP. On the right, the pore forms in the core of the *c*-ring when the lipid plug is pulled out. mPTP opening dissipates the mitochondrial ∆*p* and water entries in the matrix driven by oncotic pressure.

Dysregulated mPTP opening is involved in mitochondrial dysfunctions which feature a variety of diseases such as neurological and cardiovascular disorders, type 1 diabetes [211], cancer [212], inflammatory bone diseases [213] and diseases due to the exposure to contaminants [214]. In general, pathological conditions associated with oxidative stress also involve mPTP dysregulation [182]. Notably, the mPTP has been also involved in lifespan [215] and bone repair [216]. So, the discovery and design of mPTP rulers is at present a great challenge in pharmacology [217–219]. Recently, the use of natural products such as mPTP modulators raised a great interest in ethnomedicine [220].
