Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria

Herick, Klaus; Krämer, Reinhard; Lühring, Hinrich

doi:10.1016/s0005-2728(97)00050-9

Cited by 42 publications

(30 citation statements)

References 32 publications

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“…This is an important point because the full conductance of VDAC is similar to that of the MMC [60,61]. On the other hand, in the presence of Ca 2+ and Mg 2+ the PiC from S. cerevisiae displays channel activity with mean conductance of only 25±5 pS in 100 mM KCl [62]. Whether the low conductances we observe are due to substates of the PTP/MMC or to the presence of the PiC remains an open question.…”

Section: +mentioning

confidence: 63%

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

Carraro

Checchetto

Sartori

et al. 2018

Cell Physiol Biochem

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Background/Aims: The permeability transition pore (PTP) is an unselective, Ca²⁺-dependent high conductance channel of the inner mitochondrial membrane whose molecular identity has long remained a mystery. The most recent hypothesis is that pore formation involves the F-ATP synthase, which consistently generates Ca²⁺-activated channels. Available structures do not display obvious features that can accommodate a channel; thus, how the pore can form and whether its activity can be entirely assigned to F-ATP synthase is the matter of debate. In this study, we investigated the role of F-ATP synthase subunits e, g and b in PTP formation. Methods: Yeast null mutants for e, g and the first transmembrane (TM) α-helix of subunit b were generated and evaluated for mitochondrial morphology (electron microscopy), membrane potential (Rhodamine123 fluorescence) and respiration (Clark electrode). Homoplasmic C23S mutant of subunit a was generated by in vitro mutagenesis followed by biolistic transformation. F-ATP synthase assembly was evaluated by BN-PAGE analysis. Cu²⁺ treatment was used to induce the formation of F-ATP synthase dimers in the absence of e and g subunits. The electrophysiological properties of F-ATP synthase were assessed in planar lipid bilayers. Results: Null mutants for the subunits e and g display dimer formation upon Cu²⁺ treatment and show PTP-dependent mitochondrial Ca²⁺ release but not swelling. Cu²⁺ treatment causes formation of disulfide bridges between Cys23 of subunits a that stabilize dimers in absence of e and g subunits and favors the open state of wild-type F-ATP synthase channels. Absence of e and g subunits decreases conductance of the F-ATP synthase channel about tenfold. Ablation of the first TM of subunit b, which creates a distinct lateral domain with e and g, further affected channel activity. Conclusion: F-ATP synthase e, g and b subunits create a domain within the membrane that is critical for the generation of the high-conductance channel, thus is a prime candidate for PTP formation. Subunits e and g are only present in eukaryotes and may have evolved to confer this novel function to F-ATP synthase.

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Section: +mentioning

confidence: 63%

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

Carraro

Checchetto

Sartori

et al. 2018

Cell Physiol Biochem

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“…5), and it can be inhibited only by ADP and bongkrekic acid together, whereas ADP alone had a marginal effect (35)(36)(37). Patch-clamp experiments with the reconstituted, functionally active mitochondrial Pi carrier revealed an anion channel function with a mean conductance as low as 40 ± 10 pS, which was decreased to 25 ± 5 pS by Ca 2+ and Mg 2+ , inhibited by Pi, and unaffected by ADP (38). These features make the Pi carrier a very unlikely candidate as a PTP component, and they rule out that the currents observed here may be related to this protein.…”

Section: Discussionmentioning

confidence: 96%

Dimers of mitochondrial ATP synthase form the permeability transition pore

Giorgio

Stockum

Antoniel³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

835

882

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Here we define the molecular nature of the mitochondrial permeability transition pore (PTP), a key effector of cell death. The PTP is regulated by matrix cyclophilin D (CyPD), which also binds the lateral stalk of the F O F 1 ATP synthase. We show that CyPD binds the oligomycin sensitivity-conferring protein subunit of the enzyme at the same site as the ATP synthase inhibitor benzodiazepine 423 (Bz-423), that Bz-423 sensitizes the PTP to Ca 2+ like CyPD itself, and that decreasing oligomycin sensitivity-conferring protein expression by RNAi increases the sensitivity of the PTP to Ca 2+ . Purified dimers of the ATP synthase, which did not contain voltage-dependent anion channel or adenine nucleotide translocator, were reconstituted into lipid bilayers. In the presence of Ca 2+ , addition of Bz-423 triggered opening of a channel with currents that were typical of the mitochondrial megachannel, which is the PTP electrophysiological equivalent. Channel openings were inhibited by the ATP synthase inhibitor AMP-PNP (γ-imino ATP, a nonhydrolyzable ATP analog) and Mg 2+ /ADP. These results indicate that the PTP forms from dimers of the ATP synthase.

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“…SDH and PIC, among several other proteins, associate with mABC1 according to this technique (see Supporting Text). The identification of mABC1 as part of this complex, and reports (23)(24)(25) that PIC and ANT can exhibit ion channel activity, prompted us to evaluate the SDH-mABC1-PIC-ANT-ATPase complex for mitoK ATP activity.…”

Section: Five Mitochondrial Membrane Proteins Interact To Form a Compmentioning

confidence: 99%

Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K ⁺ channel activity

Ardehali

Chen

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

206

161

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The mitochondrial ATP-sensitive K ؉ (mitoKATP) channel plays a central role in protection of cardiac and neuronal cells against ischemia and apoptosis, but its molecular structure is unknown. Succinate dehydrogenase (SDH) is inhibited by mitoK ATP activators, fueling the contrary view that SDH, rather than mitoK ATP, is the target of cardioprotective drugs. Here, we report that SDH forms part of mitoK ATP functionally and structurally. Four mitochondrial proteins [mitochondrial ATP-binding cassette protein 1 (mABC1), phosphate carrier, adenine nucleotide translocator, and ATP synthase] associate with SDH. A purified IM fraction containing these proteins was reconstituted into proteoliposomes and lipid bilayers and shown to confer mitoK ATP channel activity. This channel activity is sensitive not only to mitoK ATP activators and blockers but also to SDH inhibitors. These results reconcile the controversy over the basis of ischemic preconditioning by demonstrating that SDH is a component of mitoK ATP as part of a macromolecular supercomplex. The findings also provide a tangible clue as to the structural basis of mitoKATP channels.A lthough prolonged ischemia results in severe damage to myocardial and neuronal cells and ultimately cell death, brief ischemic episodes can recruit a mechanism that can protect against future ischemic insults. The identification of this endogenous autoprotective mechanism, known as ischemic preconditioning (IPC), has sparked considerable interest given its potential to mitigate cellular injury in heart attack and stroke (1-3). The molecular basis of IPC remains unclear. Recent evidence suggests that the mitochondrial ATP-sensitive K ϩ channel (mitoK ATP ) is a key player in the process of IPC and is an inhibitor of apoptosis.The mitoK ATP channel was first identified in 1991 from single-channel recordings of the mitochondrial inner membrane (IM) (4). Subsequent studies (5, 6) revealed that the antihypertensive agent diazoxide is an agonist of mitoK ATP channels and protects against cardiac cell death by a mechanism like that of IPC but without the need for conditioning ischemia. These observations led to the notion that mitoK ATP channels protect against ischemia by inhibiting apoptosis and necrosis, possibly as a consequence of altered mitochondrial K ϩ or Ca 2ϩ homeostasis (7-9). Nevertheless, the existence of mitoK ATP channels has been questioned (10, 11). Inhibitors of the Krebs cycle enzyme succinate dehydrogenase (SDH) have been shown to mimic the process of IPC (12). Furthermore, some drugs that activate mitoK ATP channels also inhibit SDH (13), leading to the alternative hypothesis that respiratory inhibition, rather than channel activity, underlies IPC (10). The controversy has been fostered by the lack of a molecular identity for mitoK ATP channels. Such ignorance has also stymied rational drug development of compounds to prevent or delay ischemic injury.Given the functional and pharmacological overlap between SDH and mitoK ATP channels, we hypothesized that SDH either intera...

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Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria

Cited by 42 publications

References 32 publications

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

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

Dimers of mitochondrial ATP synthase form the permeability transition pore

Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K ⁺ channel activity

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Patch clamp investigation into the phosphate carrier from Saccharomyces cerevisiae mitochondria

Cited by 42 publications

References 32 publications

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

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

Dimers of mitochondrial ATP synthase form the permeability transition pore

Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K + channel activity

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Multiprotein complex containing succinate dehydrogenase confers mitochondrial ATP-sensitive K ⁺ channel activity