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

Significance

Mitochondria generate the cellular fuel ATP to sustain complex life. Production of ATP depends on the oxidation of energy-rich compounds to produce a chemical potential difference for hydrogen ions, the proton motive force (pmf), across the inner mitochondrial membrane (IMM). Disruption of the IMM, dissipation of the pmf, and cell death occur if the concentration of calcium ions inside mitochondria is sufficiently elevated to open a pore in the IMM. The identity of the pore is disputed. One proposal is that the pore is in the enzyme that makes ATP. Here, we show that proteins in the enzyme’s peripheral stalk are not involved in the formation or regulation of the pore.

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 insulin-sensitive tissues (DAPIT), and the 6.8-kDa proteolipid.