What is the mitochondrial permeability transition pore?

Abstract

Under conditions of mitochondrial calcium overload, especially when accompanied by oxidative stress, elevated phosphate concentrations and adenine nucleotide depletion, a non-specific pore, the mitochondrial permeability transition pore (MPTP), opens in the inner mitochondrial membrane. MPTP opening enables free passage into the mitochondria of molecules of < 1.5 kDa including protons. The resulting uncoupling of oxidative phosphorylation leads to ATP depletion and necrotic cell death and it is now widely recognised that MPTP opening is a major cause of reperfusion injury and an effective target for cardioprotection. The properties of the MPTP are well defined, but despite extensive research in many laboratories, its exact molecular identity remains uncertain. Knockout studies have confirmed a role for cyclophilin-D (CyP-D), probably mediated by its peptidyl-prolyl cis–trans isomerase activity facilitating a conformational change of an inner membrane protein. However, the identity of the membrane component(s) remains controversial. Knockout studies have eliminated an essential role for either the voltage dependent anion channel (VDAC) or the adenine nucleotide translocase (ANT), although a regulatory role for the ANT was confirmed. Our own studies implicate the mitochondrial phosphate carrier (PiC) in MPTP formation and are consistent with a calcium-triggered conformational change of the PiC, facilitated by CyP-D, inducing pore opening. We propose that this is enhanced by an association of the PiC with the “c” conformation of the ANT. Agents that modulate pore opening may act on either or both the PiC and the ANT. However, knockdown and reconstitution studies are awaited to confirm or refute this model.

Introduction

It was recognised more than 40 years ago that energised mitochondria exposed to high loads of calcium can undergo massive swelling that is characterised by a large decrease in light scattering and can be partially reversed by calcium chelation [1], [2]. It was originally suggested that this swelling might represent a non-specific permeabilisation of the mitochondrial inner membrane through activation of Ca-sensitive phospholipases leading to the build up of fatty acids and lysophospholipids in the membrane (see [3]). However, pioneering studies by Haworth and Hunter [4], [5] and later by Crompton et al. [6] provided evidence that the increase in permeability was the result of the opening of a non-specific channel of defined diameter in the membrane that exhibited a molecular cut-off of about 1.5 kDa. This channel became known at the mitochondrial permeability transition pore (MPTP) and a wide range of activators and inhibitors of pore opening were identified. These are summarised in Table 1 and will be further discussed in Section 2. Of particular significance was the observation that opening of the MPTP is greatly enhanced by adenine nucleotide depletion, elevated phosphate and oxidative stress which are conditions known to accompany reperfusion of the heart following a period of ischemia (see [7], [8], [9]).

One major consequence of MPTP opening is that the inner mitochondrial membrane no longer maintains a barrier to protons which leads to dissipation of the proton motive force. The resulting uncoupling of oxidative phosphorylation not only prevents mitochondria from making ATP, but the proton-translocating ATPase goes into reverse. This means that within any cell the “open” mitochondria hydrolyse ATP generated by glycolysis and any “closed” mitochondria, leading to ATP depletion and bioenergetic failure of the myocyte (see [7], [8]). A second consequence of MPTP opening is that all small molecular weight molecules equilibrate across the inner membrane, including cofactors and ions. This will not only lead to the disruption of metabolic gradients between the mitochondria and cytosol, including the release of accumulated Ca²⁺, but will also cause mitochondrial swelling. This occurs because the increased permeability of the inner mitochondrial membrane to small molecules mediates equilibration of all low molecular weight osmolytes whilst retaining proteins within their respective compartments. Since the matrix protein concentration is higher than that in the cytosol and intermembrane space, it exerts a colloidal osmotic pressure leading to swelling of the matrix compartment (see [7], [8], [10]). Swelling can occur without inner membrane rupture because the cristae unfold, but as the matrix expands it exerts pressure on the outer membrane that eventually ruptures. This releases cytochrome c[11], [12], [13] and other pro-apoptotic proteins which have the potential to initiate apoptotic cell death (see [14], [15], [16]). However, only if the pores close again sufficiently to maintain ATP levels will apoptopic death predominate over necrotic cell death (see [8]).

It was first was recognised by Martin Crompton that opening of the pore might account for the damage experienced by the heart during reperfusion after a period of ischemia [6], [17]. Subsequent studies in his laboratory using isolated cardiac myocytes [18], [19] and this laboratory using Langendorf perfused hearts [20], [21] demonstrated directly that MPTP opening does occur in such reperfusion injury, and that preventing pore opening provides protection against reperfusion injury. These pioneering studies were slow to be appreciated. However, in recent years data have accumulated from many different laboratories that have confirmed the central role of MPTP opening in reperfusion injury and its importance as a pharmacological target for cardioprotection (see [8], [9], [22], [23], [24]).

The role, if any, of the MPTP in healthy cells remains unclear since mice lacking cyclophilin-D (CyP-D), a component of the MPTP (see Section 3.1), appear normal but as predicted are protected against ischemic injury [25], [26], [27], [28]. However, a recent paper has reported that CyP-D knockout mice display greater anxiety with less tendency to explore and a facilitation of avoidance behaviour. They also exhibit an abnormal accumulation of white adipose tissue resulting in adult-onset obesity but whether or not the loss of MPTP opening accounts for these effects is not known [29]. Another suggested role for the MPTP in healthy cells is that it might provide a mechanism by which aging mitochondria are removed from the cell by autophagy [30]. Thus, as mitochondria age they are exposed to more oxidative stress and so become more susceptible to MPTP opening. Eventually, a time will come when the amount of oxidative stress in an older mitochondrion is sufficient to cause MPTP opening at resting calcium concentrations and this might enable it to be recognised by autophagic vacuoles and removed. Such a mechanism would prevent damaged mitochondria from proliferating whilst healthy mitochondria would continue to replicate. However, although this is an attractive hypothesis, the CyP-D knockout mice provide no direct evidence for it [29].

In view of its central role in reperfusion injury, the MPTP has become an obvious target for cardioprotection. Indeed, it has been demonstrated that a wide variety of cardioprotective protocols prevent MPTP opening during reperfusion. These include drugs that directly inhibit the MPTP such as sanglifehrin A (SfA), cyclosporin A (CsA) and its non-immunosuppressive analogues 6-MeAla-CsA, 4-methyl-val-CsA, N-methyl-4-isoleucine-CsA (NIM811) and d-3-MeAla-4-EtVal-CsA (Debio-025) [20], [21], [31], [32], [33] as well as protocols that inhibit MPTP opening indirectly through decreasing oxidative stress or pH. Such is the case for ischemic preconditioning [34] and post-conditioning [35], temperature preconditioning [36], urocortin [37], Na⁺/H⁺ exchanger inhibitors such as cariporide [38], antioxidants including pyruvate [39] and the anaesthetic propofol [40] and mitochondrial-targeted ubiquinone antioxidants [41].

A drug that inhibits the MPTP directly has the potential to be of great value in protecting the heart during cardiac surgery or the treatment of a coronary thrombosis. Indeed, proof of principal has been provided by preliminary studies demonstrating that CsA improved cardiac performance following treatment of coronary thrombosis with angioplasty [42]. However, CsA and Sfa are not ideal for cardioprotection for two reasons. First, they have the potential to exert unwanted side-effects through their interactions with other cyclophilins such as Cyp-A (see [8]). Second, their ability to inhibit MPTP opening is overcome as the magnitude of the stimulus responsible for MPTP opening ([Ca²⁺], oxidative stress or adenine nucleotide depletion) is increased [21], [31], [43], [44]. Thus it would be desirable to develop new drugs that target the MPTP whose potency is not constrained in this way. A major obstacle that stands in the way of achieving this goal is our uncertainty over the exact molecular composition and mechanism of the MPTP. In this review I will attempt to summarise what the current state of knowledge is.