Andrew P. Halestrap

Mitochondrial permeability transition pore opening during myocardial reperfusion: a target for cardioprotection

Reperfusion of the heart after a period of ischaemia leads to the opening of a nonspecific pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (MPTP). This transition causes mitochondria to become uncoupled and capable of hydrolysing rather than synthesising ATP. Unrestrained, this will lead to the loss of ionic homeostasis and ultimately necrotic cell death. The functional recovery of the Langendorff-perfused heart from ischaemia inversely correlates with the extent of pore opening, and inhibition of the MPTP provides protection against reperfusion injury. This may be mediated either by a direct interaction with the MPTP [e.g., by Cyclosporin A (CsA) and Sanglifehrin A (SfA)], or indirectly by decreasing calcium loading and reactive oxygen species (ROS; key inducers of pore opening) or lowering intracellular pH. Agents working in this way may include pyruvate, propofol, Na+/H+ antiporter inhibitors, and ischaemic preconditioning (IPC). Mitochondrial KATP channels have been implicated in preconditioning, but our own data suggest that the channel openers and blockers used in these studies work through alternative mechanisms. In addition to its role in necrosis, transient opening of the MPTP may occur and lead to the release of cytochrome c and other proapoptotic molecules that initiate the apoptotic cascade. However, only if subsequent MPTP closure occurs will ATP levels be maintained, ensuring that cell death continues down an apoptotic, rather than a necrotic, pathway.

The SLC16 gene family—from monocarboxylate transporters (MCTs) to aromatic amino acid transporters and beyond

The monocarboxylate cotransporter (MCT) family now comprises 14 members, of which only the first four (MCT1–MCT4) have been demonstrated experimentally to catalyse the proton-linked transport of metabolically important monocarboxylates such as lactate, pyruvate and ketone bodies. SLC16A10 (T-type amino-acid transporter-1, TAT1) is an aromatic amino acid transporter whilst the other members await characterization. MCTs have 12 transmembrane domains (TMDs) with intracellular N- and C-termini and a large intracellular loop between TMDs 6 and 7. MCT1 and MCT4 require a monotopic ancillary protein, CD147, for expression of functional protein at the plasma membrane. Lactic acid transport across the plasma membrane is fundamental for the metabolism of and pH regulation of all cells, removing lactic acid produced by glycolysis and allowing uptake by those cells utilizing it for gluconeogenesis (liver and kidney) or as a respiratory fuel (heart and red muscle). The properties of the different MCT isoforms and their tissue distribution and regulation reflect these roles.

The permeability transition pore complex: another view

Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca(2+)], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl-prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca(2+) or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys(56) and Cys(159)) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys(159) modification whilst oxidation of Cys(56) increases CyP-D binding to the ANT, probably at Pro(61).

Oxidative Stress, Thiol Reagents, and Membrane Potential Modulate the Mitochondrial Permeability Transition by Affecting Nucleotide Binding to the Adenine Nucleotide Translocase

Stimulation of the mitochondrial permeability transition (MPT) in de-energized mitochondria by phenylarsine oxide (PheArs) is greater than that by diamide and t-butylhydroperoxide (TBH), yet the increase in CyP binding to the inner mitochondrial membrane (Connern, C. P. and Halestrap, A. P. (1994) Biochem. J. 302, 321-324) is less. From a range of nucleotides tested only ADP, deoxy-ADP, and ATP inhibited the MPT. ADP inhibition involved two sites with Ki values of about 1 and 25 μM which were independent of [Ca2+] and CyP binding. Carboxyatractyloside (CAT) abolished the high affinity site. Following pretreatment of mitochondria with TBH or diamide, the Ki for ADP increased to 50-100 μM, whereas pretreatment with PheArs or eosin maleimide increased the Ki to >500 μM; only one inhibitory site was observed in both cases. Eosin maleimide is known to attack Cys159 of the adenine nucleotide translocase (ANT) in a CAT-sensitive manner (Majima, E., Shinohara, Y., Yamaguchi, N., Hong, Y. M., and Terada, H. (1994) Biochemistry 33, 9530-9536), and here we demonstrate CAT-sensitive binding of the ANT to a PheArs affinity column. In adenine nucleotide-depleted mitochondria, no stimulation of the MPT by uncoupler was observed in the presence or absence of thiol reagents, suggesting that membrane potential may inhibit the MPT by increasing adenine nucleotide binding through an effect on the ANT conformation. We conclude that CsA and ADP inhibit pore opening in distinct ways, CsA by displacing bound CyP and ADP by binding to the ANT. Both mechanisms act to decrease the Ca2+ sensitivity of the pore. Thiol reagents and oxidative stress may modify two thiol groups on the ANT and thus stimulate pore opening by both means.

Identification of Monocarboxylate Transporter 8 as a Specific Thyroid Hormone Transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an ∼10-fold increase in uptake of 10 nm 125I-labeled thyroxine (T4), 3,3′,5-triiodothyronine (T3), 3,3′,5′-triiodothyronine (rT3) and 3,3′-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by l-T4, d-T4, l-T3, d-T3, 3,3′,5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 μm for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.

Association between mutations in a thyroid hormone transporter and severe X-linked psychomotor retardation

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transporter, the gene of which is located on the X chromosome. We tested whether mutations in MCT8 cause severe psychomotor retardation and high serum triiodothyronine (T3) concentrations in five unrelated young boys. The coding sequence of MCT8 was analysed by PCR and direct sequencing of its six exons. In two patients, gene deletions of 2.4 kb and 24 kb were recorded and in three patients missense mutations Ala150Val, Arg171 stop, and Leu397Pro were identified. We suggest that this novel syndrome of X-linked psychomotor retardation is due to a defect in T3 entry into neurons through MCT8, resulting in impaired T3 action and metabolism.

The Plasma Membrane Lactate Transporter MCT4, but Not MCT1, Is Up-regulated by Hypoxia through a HIF-1α-dependent Mechanism

The monocarboxylate transporter MCT4 mediates lactic acid efflux from most tissues that are dependent on glycolysis for their ATP production. Here we demonstrate that expression of MCT4 mRNA and protein was increased >3-fold by a 48-h exposure to 1% O2, whereas MCT1 expression was not increased. The effect was mimicked by CoCl2 (50 μm), suggesting transcriptional regulation by hypoxia-inducible factor 1α (HIF-1α). The predicted promoters for human MCT1, MCT2, and MCT4 were cloned into the pGL3 vector and shown to be active (luciferase luminescence) under basal conditions. Only the MCT4 promoter was activated (>2-fold) by hypoxia. No response was found in cells lacking HIF-1α. Four potential hypoxia-response elements were identified, but deletion analysis implicated only two in the hypoxia response. These were just upstream from the transcription start site and also found in the mouse MCT4 promoter. Mutation of site 2 totally abolished the hypoxic response, whereas mutation of site 1 only reduced the response. Gel-shift analysis demonstrated that nuclear extracts of hypoxic but not normoxic HeLa cells contained two transcription factors that bound to DNA probes containing these hypoxia-response elements. The major shifted band was abolished by mutation of site 2, and supershift analysis confirmed that HIF-1α bound to this site. Binding of the second factor was abolished by mutation of site 1. We conclude that MCT4, like other glycolytic enzymes, is up-regulated by hypoxia through a HIF-1α-mediated mechanism. This adaptive response allows the increased lactic acid produced during hypoxia to be rapidly lost from the cell.

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin‐like domains and a single charge‐containing transmembrane domain. Here we use co‐immunoprecipitation and chemical cross‐linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton‐linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2–CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co‐localize, concentrating at the t‐tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross‐linking with anti‐CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into ‘caps’. In MCT‐transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co‐transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.

Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart

First, we present a summary of the evidence for our model of the molecular mechanism of the permeability transition (MPT). Our proposal is that the MPT occurs as a result of the binding of mitochondrial cyclophilin (CyP-D) to the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane. This binding is enhanced by thiol modification of the ANT caused by oxidative stress or other thiol reagents. CyP-D binding enhances the ability of the ANT to undergo a conformational change triggered by Ca2+. Binding of ADP or ATP to a matrix site of the ANT antagonises this effect of Ca2+; modification of other ANT thiol groups inhibits ADP binding and sensitises the MPT to [Ca2+]. Increased membrane potential changes the ANT conformation to enhance ATP binding and hence inhibit the MPT. Our most recent data shows that a fusion protein of CyP-D and glutathione-S-transferase immobilised to Sepharose specifically binds the ANT from Triton-solubilised inner mitochondrial membranes in a cyclosporin A (CsA) sensitive manner. Second we summarise the evidence for the MPT being a major factor in the transition from reversible to irreversible injury during reperfusion of a heart following a period of ischaemia. We describe how in the perfused heart [3H]deoxyglucose entrapment within mitochondria can be used to measure the opening of MPT pore in situ. During ischaemia pore opening does not occur, but significant opening does occur during reperfusion, and recovery of the heart is dependent on subsequent pore closure. Pore opening is inhibited by the presence in the perfusion medium of pyruvate and the anaesthetic propofol which both protect the heart from reperfusion injury. Third we discuss how the MPT may be involved in determining whether cell death occurs by necrosis (extensive pore opening and ATP depletion) or apoptosis (transient pore opening with maintenance of ATP).

Calcium, mitochondria and reperfusion injury: a pore way to die

When mitochondria are exposed to high Ca2+ concentrations, especially when accompanied by oxidative stress and adenine nucleotide depletion, they undergo massive swelling and become uncoupled. This occurs as a result of the opening of a non-specific pore in the inner mitochondrial membrane, known as the MPTP (mitochondrial permeability transition pore). If the pore remains open, cells cannot maintain their ATP levels and this will lead to cell death by necrosis. This article briefly reviews what is known of the molecular mechanism of the MPTP and its role in causing the necrotic cell death of the heart and brain that occurs during reperfusion after a long period of ischaemia. Such reperfusion injury is a major problem during cardiac surgery and in the treatment of coronary thrombosis and stroke. Prevention of MPTP opening either directly, using agents such as cyclosporin A, or indirectly by reducing oxidative stress or Ca2+ overload, provides a protective strategy against reperfusion injury. Furthermore, mice in which a component of the MPTP, CyP-D (cyclophilin D), has been knocked out are protected against heart and brain ischaemia/reperfusion. When cells experience a less severe insult, the MPTP may open transiently. The resulting mitochondrial swelling may be sufficient to cause release of cytochrome c and activation of the apoptotic pathway rather than necrosis. However, the CyP-D-knockout mice develop normally and show no protection against a range of apoptotic stimuli, suggesting that the MPTP does not play a role in most forms of apoptosis.