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THE ATP SYNTHASE AND THE PERMEABILITY TRANSITION IN MITOCHONDRIA

The permeability transition in human mitochondria refers to the opening of a non-specific channel, known as the permeability transition pore (PTP) in the inner membrane. Opening can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, followed by cell death [1] [2]. These events have been linked to pathways leading to cell death, and to human diseases including cardiac ischemia and muscle dystrophy [3]. The opening of the pore can be induced artificially by compounds such as thapsigargin [4], a non-competitive inhibitor of the Ca2+-ATPase in the sarcoplasmic and endoplasmic reticula, and by ionophores for divalent cations such as ionomycin [5], and ferutinin [6]. It can also be inhibited by drugs such as cyclosporin A, mediated via its binding to the prolyl cis-trans isomerase, cyclophilin D, in the mitochondrial matrix [7] [8]. Many proposals have been made about the protein constituents of the PTP itself, including the ADP/ATP translocase, an abundant component of the inner membranes of mitochondria, and the voltage dependent anion channel found in the outer membranes of the organelle, but none of these proposals has been established definitively [9] [10]. A further proposition, that another component of the inner mitochondrial membrane, the AAA-protease, SPG7, participates in formation of the pore, has been disputed [11] [12].

Recently, it has been proposed that the PTP is associated with the ATP synthase complex [13]. Some proposals suggest that the ring of c-subunits that constitute the membrane domain of the enzyme’s rotor provides the pore [14] [15] [16]. Others suggest that the pore is associated with membrane subunits of the enzyme found in the region of interface between monomers in the dimeric ATP synthase complexes found in mitochondria [13] [17].
 

  • We have tested the proposal that the PTP is associated with the c-subunit by producing a clonal human cell line where all three genes encoding subunit c have been disrupted. The cells are devoid of subunit c, and yet the characteristic properties of the PTP persist. Therefore, the c8-ring of human ATP synthase does not provide the PTP [18].

 

  • We have also examined ρ0 human cells for the presence of the PTP. These cells have no mitochondrial DNA, and therefore the ATP6 and ATP8 membrane subunits of the ATP synthase are absent from their mitochondria. The PTP persists in these cells also. Therefore, the ATP6 and ATP8 subunits do not provide the PTP [18].

 

  • By disrupting the human genes encoding the OSCP and subunit b, we have shown that the permeability transition is unaffected by the absence of the peripheral stalk [19]. This is a highly significant finding as according to work published by others, the OSCP provides the site with which cyclophilin D interacts [13]. Moreover, more recently published work suggests that Ca2+ ions bind in the region of the catalytic sites of the ATPase, and that their effect is transmitted to the pore in membrane domain via the peripheral stalk [20] [21] [22] [23]. These proposals are no longer tenable.

 

  • We have disrupted individually the human genes for the supernumerary subunits e, f, g, 6.8 proteolipid and DAPIT (diabetes associated protein in insulin sensitive issue), and in each case characterized the vestigial ATPase complex, and examined the effect of each deletion on the PTP. Disruption of each of these genes has no effect on the PTP [24].

 

  • In the human cell line where the three genes for subunit c had been disrupted, we have also disrupted the gene for the δ-subunit, a component of the F1-catalytic domain. The mitochondria of these cells lack an assembled ATPase complex. The only remaining vestige is a sub-complex of the peripheral stalk, containing subunit b, e and g. Yet, despite the diminished levels of respiratory complexes I, III and IV in these mitochondria, they still generate a membrane potential sufficient to drive the uptake of exogeneous Ca2+, and they retain a fully functional PTP with all of its characteristic properties intact [24].

 

  • On the basis of these extensive investigations, we conclude that the proposal that the dimeric ATPase complex provides the PTP is not tenable.

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