Effect of anions on Cyclophilin D binding to F-ATP synthase: Implications for the permeability transition pore

The c subunit of the ATP synthase is an inner mitochondrial membrane (IMM) protein. Besides its role as the main component of the rotor of the ATP synthase, c subunit from mammalian mitochondria exhibits ion channel activity. In particular, c subunit may be involved in one of the pathways leading to the formation of the permeability transition pore (PTP) during mitochondrial permeability transition (PT), a phenomenon consisting of the permeabilization of the IMM due to high levels of calcium. Our previous study on the synthetic c subunit showed that high concentrations of calcium induce misfolding into cross-β oligomers that form low-conductance channels in model lipid bilayers of about 400 pS. Here, we studied the effect of cyclophilin D (CypD), a mitochondrial chaperone and major regulator of PTP, on the electrophysiological activity of the c subunit to evaluate its role in the functional properties of c subunit. Our study shows that in presence of CypD, c subunit exhibits a larger conductance, up to 4 nS, that could be related to its potential role in mitochondrial toxicity. Further, our results suggest that CypD is necessary for the formation of c subunit induced PTP but may not be an integral part of the pore.

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TRAP1 and cyclophilin D compete at OSCP subunit to regulate enzymatic activity and permeability transition pore opening by F-ATP synthase

10.1101/2021.12.06.471412 ◽

2021 ◽

Author(s):

Giuseppe Cannino ◽

Andrea Urbani ◽

Marco Gaspari ◽

Mariaconcetta Varano ◽

Alessandro Negro ◽

...

Keyword(s):

Atp Synthase ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Inhibitory Effect ◽

Cyclophilin D ◽

Electrophysiological Measurements ◽

Permeability Transition Pore Opening ◽

Pore Opening ◽

Client Proteins ◽

Oligomycin Sensitivity Conferring Protein

AbstractBinding of the mitochondrial chaperone TRAP1 to client proteins shapes cell bioenergetic and proteostatic adaptations, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Moreover, TRAP1 inhibits opening of the permeability transition pore (PTP) formed by F-ATP synthase and effectively antagonizes the PTP-inducing effect of CyPD, which elicits mitochondrial depolarization and cell death. Consistently, electrophysiological measurements indicate that TRAP1 and CyPD compete in the modulation of channel activity of purified F-ATP synthase, resulting in PTP inhibition and activation, respectively, and outcompeting each other effect on the channel. Moreover, TRAP1 counteracts PTP induction by CyPD, whereas CyPD reverses TRAP1-mediated PTP inhibition. Our data identify TRAP1 as a F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711201114 ◽

2017 ◽

Vol 114 (34) ◽

pp. 9086-9091 ◽

Cited By ~ 99

Author(s):

Jiuya He ◽

Joe Carroll ◽

Shujing Ding ◽

Ian M. Fearnley ◽

John E. Walker

Keyword(s):

Atp Synthase ◽

Catalytic Domain ◽

Atp Synthesis ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Helical Structure ◽

Cyclophilin D ◽

Membrane Domain ◽

Peripheral Stalk ◽

Subunit B

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.

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Abstract 331: Impact of Acetylated Cyclophilin D on the Mitochondrial Permeability Transition Pore

Circulation Research ◽

10.1161/res.127.suppl_1.331 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Gisela Beutner ◽

Jacob Perkins ◽

Ronak A Sardari ◽

George A Porter

Keyword(s):

Atp Synthase ◽

Cyclosporine A ◽

Matrix Protein ◽

Mitochondrial Permeability Transition ◽

Atp Synthesis ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Cyclophilin D ◽

Retention Capacity ◽

Transport Chain

Background: The mitochondrial matrix protein cyclophilin D (CypD) is a key regulator of mitochondrial function. CypD controls electron transport chain activity and ATP synthesis by regulating the permeability transition pore (PTP). The activity of CypD is regulated by several post-translational modifications including acetylation of lysine 166 in the mouse. Objective: To investigate how acetylation at lysine 166 of CypD specifically in the heart modifies its ability to regulate the PTP and the ATP synthase. Results: We generated a conditional cardiac knock-in mouse model where lysine 166 has been mutated into glutamine (CypD K166Q ) to mimic permanent acetylation of CypD. The mice were either +/+, +/- or -/- for the expression of native CypD. Results show that mitochondrial oxygen consumption was not affected by the expression of CypD K166Q . The calcium retention capacity (CRC) was measured with Arsenazo III and decreased significantly when CypD K166Q was expressed. The CypD inhibitor cyclosporine A significantly increased the CRC in WT mice. However, cyclosporine A was did not inhibit CypD in the hearts of mice expressing only CypD K166Q or in addition to wild-type CypD. The ability of the ATP synthase to create dimers or oligomers was assessed by western blotting and the hydrolysis of ATP in in-gel assays and shows that expression of CypD K166Q decreased the assembly of the ATP synthase into dimers or oligomers. Conclusions: Our data show that the expression of CypD K166Q increases the sensitivity of PTP opening to calcium and limits the assembly of ATP synthase into oligomers.

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Persistence of the permeability transition pore in human mitochondria devoid of an assembled ATP synthase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904005116 ◽

2019 ◽

Vol 116 (26) ◽

pp. 12816-12821 ◽

Cited By ~ 45

Author(s):

Joe Carroll ◽

Jiuya He ◽

Shujing Ding ◽

Ian M. Fearnley ◽

John E. Walker

Keyword(s):

Atp Synthase ◽

Catalytic Domain ◽

Total Concentration ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Cyclophilin D ◽

Peripheral Stalk ◽

Associated Proteins ◽

Pore Opening ◽

Membrane Bound

The opening of the permeability transition pore, a nonspecific channel in inner mitochondrial membranes, is triggered by an elevated total concentration of calcium ions in the mitochondrial matrix, leading to disruption of the inner membrane and necrotic cell death. Cyclosporin A inhibits pore opening by binding to cyclophilin D, which interacts with the pore. It has been proposed that the pore is associated with the ATP synthase complex. Previously, we confirmed an earlier observation that the pore survives in cells lacking membrane subunits ATP6 and ATP8 of ATP synthase, and in other cells lacking the enzyme’s c8rotor ring or, separately, its peripheral stalk subunits b and oligomycin sensitive conferral protein. Here, we investigated whether the pore is associated with the remaining membrane subunits of the enzyme. Individual deletion of subunits e, f, g, and 6.8-kDa proteolipid disrupts dimerization of the complex, and deletion of DAPIT (diabetes-associated protein in insulin sensitive tissue) possibly influences oligomerization of dimers, but removal of each subunit had no effect on the pore. Also, we removed together the enzyme’s membrane bound c8ring and the δ-subunit from the catalytic domain. The resulting cells assemble only a subcomplex derived from the peripheral stalk and membrane-associated proteins. Despite diminished levels of respiratory complexes, these cells generate a membrane potential to support uptake of calcium into the mitochondria, leading to pore opening, and retention of its characteristic properties. It is most unlikely that the ATP synthase, dimer or monomer, or any component, provides the permeability transition pore.

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Faculty Opinions recommendation of Cysteine 203 of cyclophilin D is critical for cyclophilin D activation of the mitochondrial permeability transition pore.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717968331.793468042 ◽

2012 ◽

Author(s):

Heinz Osiewacz

Keyword(s):

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Cyclophilin D ◽

Mitochondrial Permeability

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Faculty Opinions recommendation of Dimers of mitochondrial ATP synthase form the permeability transition pore.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718013284.793478290 ◽

2013 ◽

Author(s):

David Criddle ◽

Muhammad Ahsan Javed

Keyword(s):

Atp Synthase ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mitochondrial Atp Synthase

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Abstract 168: Resveratrol Translocates Gsk-3β To Mitochondria And Protects The Heart At Reperfusion By Targeting The Mitochondrial Permeability Transition Pore

Circulation ◽

10.1161/circ.116.suppl_16.ii_11-b ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Jinkun Xi ◽

Huihua Wang ◽

Guillaume Chanoit ◽

Guang Cheng ◽

Robert A Mueller ◽

...

Keyword(s):

Reperfusion Injury ◽

Mitochondrial Permeability Transition Pore ◽

Mitochondrial Permeability Transition ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Cyclophilin D ◽

Risk Zone ◽

Mitochondrial Permeability ◽

Mptp Opening ◽

Gsk 3Β

Although resveratrol has been demonstrated to be cardioprotective, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. We aimed to determine if resveratrol protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β). Resveratrol (10μM) given at reperfusion reduced infarct size (12.2 ± 2.5 % of risk zone vs. 37.9 ± 3.1 % of risk zone in control, n = 6) in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion, an effect that was abrogated by the mPTP opener atractyloside (30.9 ± 8.1 % of risk zone), implying that resveratrol may protect the heart at reperfusion by modulating the mPTP opening. To define the signaling mechanism underlying the action of resveratrol, we determined GSK-3β activity by measuring its phosphorylation at Ser 9 . Resveratrol significantly enhanced GSK-3β phosphorylation upon reperfusion (225.2 ± 30.0 % of control at 5 min of reperfusion). Further experiments showed that resveratrol induces translocation of GSK-3β to mitochondria and translocated GSK-3β interacts with the mPTP component cyclophilin D but not VDAC (the voltage-dependent anion channel) or ANT (the adenine nucleotide translocator) in cardiac mitochondria. Taken together, these data suggest that resveratrol prevents myocardial reperfusion injury by targeting the mPTP opening via GSK-3β. Translocation of GSK-3β to mitochondria and its interaction with the mPTP component cyclophilin D may serve as an essential mechanism that mediates the protective effect of resveratrol on reperfusion injury.

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