Permeability transition in human mitochondria persists in the absence of peripheral stalk subunits of ATP synthase

Abstract

The opening of a nonspecific channel, known as the permeability transition pore (PTP), in the inner membranes of mitochondria can be triggered by calcium ions, leading to swelling of the organelle, disruption of the inner membrane and ATP synthesis, and cell death. Pore opening can be inhibited by cyclosporin A mediated via cyclophilin D. It has been proposed that the pore is associated with the dimeric ATP synthase and the oligomycin sensitivity conferral protein (OSCP), a component of the enzyme's peripheral stalk, provides the site at which cyclophilin D interacts. Subunit b contributes a central α-helical structure to the peripheral stalk, extending from near the top of the enzyme's catalytic domain and crossing the membrane domain of the enzyme via two α-helices. We investigated the possible involvement of the subunit b and the OSCP in the PTP by generating clonal cells, HAP1-Δb and HAP1-ΔOSCP, lacking the membrane domain of subunit b or the OSCP, respectively, in which the corresponding genes, ATP5F1 and ATP5O, had been disrupted. Both cell lines preserve the characteristic properties of the PTP; therefore, the membrane domain of subunit b does not contribute to the PTP, and the OSCP does not provide the site of interaction with cyclophilin D. The membrane subunits ATP6, ATP8, and subunit c have been eliminated previously from possible participation in the PTP; thus, the only subunits of ATP synthase that could participate in pore formation are e, f, g, diabetes-associated protein in insulinsensitive tissues (DAPIT), and the 6.8-kDa proteolipid.human mitochondria | ATP synthase | permeability transition pore | ATP5F1 subunit b | ATP5O oligomycin sensitivity conferral protein

High-Conductance Channel Formation in Yeast Mitochondria is Mediated by F-ATP Synthase e and g Subunits

“…This observation is important because it demonstrates that lack of swelling cannot be explained by ultrastructural differences in cristae and matrix morphology. As recently noted [47] these data are reminiscent of the effects of deletion of subunits b and OSCP in human cells [46] and suggest that the size of the channel resulting from deletion of the dimerization subunits is close to the exclusion limit of sucrose. Consistently, electron microscopy images revealed that mitochondria of ΔeΔg mutants still possess a dense matrix after PTP opening, while swelling occurred after the addition of Alamethicin (Fig.…”

“…Since pore size is affected by lack of lateral stalk subunits [46] we analyzed PTP-dependent swelling in mitochondria devoid of e and g subunits. We loaded mitochondria with a Ca 2+ bolus of the same size and triggered PTP opening with the SH reagent copper-o-phenantroline Cu(OP) 2 release readily ensued, and its rate was not substantially affected in any of the mutants (Fig.…”

“…We have suggested that the apparent discrepancies can be explained when the pore size, rather than occurrence of PTP opening as such, is considered [47]. Indeed, PTP-dependent mitochondrial swelling in KCl-based media was drastically decreased in the OSCP-and b-null cells [46] indicating that the pore size was becoming close to the exclusion size for K + or Cl -. Our current findings indicate that ablation of the e and g subunits drastically decreases the high-conductance currents associated with the PTP/MMC, with persistence of low-conductance states that are influenced by deletion of the first TM a-helix of subunit b.…”

“…On the other hand, genetic ablation of either the OSCP or b subunits [46] or of subunit c [53] led to the assembly of defective F-ATP synthase. Yet, upon cell permeabilization mitochondria underwent CsA-sensitive Ca 2+ -induced Ca 2+ release [46,53] and cobalt-dependent quenching of mitochondrially-entrapped calcein [46], both events being consistent with occurrence of PTP opening. We have suggested that the apparent discrepancies can be explained when the pore size, rather than occurrence of PTP opening as such, is considered [47].…”

F‐ATP synthase and the permeability transition pore: fewer doubts, more certainties

“…In both cases (a) a vestigial F‐ATP synthase was assembled that additionally lacked several subunits of the peripheral stalk and (b) a severe respiratory defect was present, with basal rates of about 20% of those of parental cells, lack of sensitivity to oligomycin and negligible stimulation by uncoupler. Consistently, these cell lines also displayed major alterations in respiratory chain assembly, with virtually no mature complex I, decreased complex III and lower levels of complex IV . Even more surprisingly given the absence of complex I, also in this case after cell permeabilization with digitonin mitochondria incubated with glutamate and malate accumulated Ca 2+ ; further, they underwent spontaneous release after the same Ca 2+ load and responded to CsA just as their parental, respiratory‐competent counterpart .…”