The nonspecific inner membrane pore of liver mitochondria: Modulation of cyclosporin sensitivity by ADP at carboxyatractyloside-sensitive and insensitive sites

doi:10.1016/S0006-291X(05)81250-1

Biochemical and Biophysical Research Communications

Volume 180, Issue 1, 15 October 1991, Pages 33-38

https://doi.org/10.1016/S0006-291X(05)81250-1 Get rights and content

Cyclosporin A prevents the opening of a nonspecific pore in the inner membrane of liver mitochondria when added prior to Ca²⁺. In the presence of 10 μM Ca²⁺ cyclosporin is unable to close the pore and restore the original permeability unless ADP is also added. ADP acts at a high-affinity site (K_m 5 μM), corresponding to the adenine nucleotide transporter. This effect of ADP is prevented and reversed by carboxyatractyloside. In the presence of carboxyatractyloside, cyclosporin added with higher concentrations of ADP (K_m 70 μM) also can close the pore. This suggests that a lower-affinity ADP-binding component as well as cyclophilin and the adenine nucleotide transporter can modulate the sensitivity of the pore to cyclosporin.

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Cited by (37)

Changes in the mitochondrial permeability transition pore in aging and age-associated diseases
2013, Mechanisms of Ageing and Development
Citation Excerpt :
It was originally suggested that ANT might be a core element of the MPTP complex (Halestrap and Davidson, 1990). The first evidence implicating ANT in MPTP formation came from studies in which ATP and bongkrekic acid (Bka), an inhibitor of ANT, inhibited opening of the MPTP by decreasing its sensitivity to Ca2+, while carboxyatactyloside (Cat) and adenine nucleotide depletion were able to trigger pore opening (Novgorodov et al., 1991; Halestrap et al., 1997). Although extensive data support ANT as a key component of MPTP, studies with knockout mice showed that ANT was not necessary for MPTP formation (Kokoszka et al., 2004).
Aging is a biological process associated with impairment of mitochondrial bioenergetic function, increased oxidative stress, attenuated ability to respond to stresses and increased risk in contracting age-associated diseases. When mitochondria are subjected to oxidative stress, accompanied by calcium overload and ATP depletion, they undergo “a permeability transition”, characterized by sudden induced change of the inner mitochondrial membrane permeability for water as well as for low-molecular weight solutes (≤1.5 kDa), resulting in membrane depolarization and uncoupling of oxidative phosphorylation. Research interest in the entity responsible for this phenomenon, the “mitochondrial permeability transition pore” (MPTP) has dramatically increased after demonstration that it plays a key role in the life and death decision in cells. The molecular structure and identity of MPTP is not yet known, although the pore is thought to exist as multiprotein complex. Some evidence indicate that the sensitivity of mitochondria to Ca²⁺-induced MPTP opening increases with aging; however the basis of this difference is unknown. Changes in MPTP structure and/or function may have important implications in the aging process and aged-associated diseases. This article examines data relevant to this issue. The important role of a principal lipidic counter-partner of the MPTP, cardiolipin, will also be discussed.
The mitochondrial permeability transition pore (PTP) - An example of multiple molecular exaptation?
2012, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
However, the PTP can be assembled and function even in the absence of CyP-D, which modulates the Ca2+-dependent PTP opening, with no consequences on the PTP regulation [76]. The mitochondrial PT regulation by adenine nucleotides is known since 1979 [7], but has been well elucidated only in the early 1990s [15,81]. The evidence that the PTP opening is inhibited by ATP and ADP, but not by their Mg2+ complexes or by other nucleotides (AMP, GDP, GTP) [82,83], suggests that the proper target of PTP inhibition by adenine nucleotides is ANT [44], because also bongkrekic acid (inhibitor) and carboxyatractyloside (stimulator) could set the ANT in an opposite conformation [84], affecting the pore in reverse directions.
The mitochondrial permeability transition (PT) is a well-recognized phenomenon that allows mitochondria to undergo a sudden increase of permeability to solutes with molecular mass ≤ 1500 Da, leading to organelle swelling and structural modifications. The relevance of PT relies on its master role in the manifestation of programmed cell death (PCD). This function is performed by a mega-channel (in some cases inhibited by cyclosporin A) named permeability transition pore (PTP), whose function could derive from the assembly of different mitochondrial proteins.
In this paper we examine the distribution and characteristics of PTP in mitochondria of eukaryotic organisms so far investigated in order to draw a hypothesis on the mechanism of its evolution. As a result, we suggest that PTP may have arisen as a new function linked to a multiple molecular exaptation of different mitochondrial proteins, even though they could nevertheless still play their original role.
Furthermore, we suggest that the early appearance of PTP could have had a crucial role in the establishment of endosymbiosis in eukaryotic cells, by the coordinated balancing of ATP production by glycolysis (performed by the primary phagocyte) and oxidative phosphorylation (accomplished by the endosymbiont). Indeed, we argue on the possibility that this new energetic equilibrium could have opened the way to the subsequent evolution toward metazoans.
The role of the mitochondrial permeability transition pore in heart disease
2009, Biochimica et Biophysica Acta - Bioenergetics
Citation Excerpt :
However, the ability of different ligands of the ANT and PiC to either activate or inhibit mPTP opening (see below) is not readily reconcilable with this model. Work from several laboratories, including our own, has demonstrated that opening of the mPTP can be inhibited by ATP, ADP and bongkrekic acid (BKA), an inhibitor of the ANT, all of which decreased the sensitivity of pore opening to [Ca2+] [26,30,42]. Conversely, activation is achieved by adenine nucleotide depletion or another inhibitor of the ANT, carboxyatractyloside (CAT) that sensitise the pore to [Ca2+] [40].
Like Dr. Jeckyll and Mr. Hyde, mitochondria possess two distinct persona. Under normal physiological conditions they synthesise ATP to meet the energy needs of the beating heart. Here calcium acts as a signal to balance the rate of ATP production with ATP demand. However, when the heart is overloaded with calcium, especially when this is accompanied by oxidative stress, mitochondria embrace their darker side, and induce necrotic cell death of the myocytes. This happens acutely in reperfusion injury and chronically in congestive heart failure. Here calcium overload, adenine nucleotide depletion and oxidative stress combine forces to induce the opening of a non-specific pore in the mitochondrial membrane, known as the mitochondrial permeability transition pore (mPTP). The molecular nature of the mPTP remains controversial but current evidence implicates a matrix protein, cyclophilin-D (CyP-D) and two inner membrane proteins, the adenine nucleotide translocase (ANT) and the phosphate carrier (PiC). Inhibition of mPTP opening can be achieved with inhibitors of each component, but targeting CyP-D with cyclosporin A (CsA) and its non-immunosuppressive analogues is the best described. In animal models, inhibition of mPTP opening by either CsA or genetic ablation of CyP-D provides strong protection from both reperfusion injury and congestive heart failure. This confirms the mPTP as a promising drug target in human cardiovascular disease. Indeed, the first clinical trials have shown CsA treatment improves recovery after treatment of a coronary thrombosis with angioplasty.
What is the mitochondrial permeability transition pore?
2009, Journal of Molecular and Cellular Cardiology
Citation Excerpt :
The site of the inhibitory effects of adenine nucleotides is thought to be the ANT. Thus work from several laboratories, including our own, has demonstrated that the sensitivity of the MPTP to matrix [Ca2+] opening can be greatly reduced by ATP and ADP, but not by their complexes with Mg2+ or by other nucleotides that are not transported by the ANT such as AMP, GDP or GTP [5,47,69,70]. Indeed, the potency of nucleotides as inhibitors of MPTP opening correlates with their ability to act as substrates of the ANT [47,71].
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.
Quantitative evaluation of the effects of mitochondrial permeability transition pore modifiers on accumulation of calcium phosphate: Comparison of rat liver and brain mitochondria
2004, Archives of Biochemistry and Biophysics
Citation Excerpt :
The first was CATR-sensitive and had high affinity for ADP. It is believed that this site corresponds to the ANT [36,37]. The second site had much lower affinity for ADP and was insensitive to CATR.
Mitochondria play a critical role in some forms of apoptosis, and the Ca²⁺-dependent permeability transition (PT) is a key initiator of this process. We quantitatively examined major control mechanisms of PT in rat brain (RBM) and liver (RLM) mitochondria. Compared with RLM, RBM were less sensitive to cyclosporin A (CsA), but the combined action of CsA + ADP was much more pronounced in RBM. Carboxyatractyloside abrogated the effects of all mPTP inhibitors in RBM but not in RLM, where the effects of CsA were not reduced. Estimated H⁺/Ca²⁺ ratios were 0.81 ± 0.01 for RLM and 0.84 − 0.93 for RBM, suggesting that Ca²⁺ and Pi were sequestered in the matrix as CaHPO₄ and Ca₃(PO₄)₂ salts, and that RBM sequester more CaPi as the least soluble salt. We conclude that: (1) RBM and RLM differ in their baseline behavior of the PT and in their responses to PT modifiers, and (2) PT modifiers can be functionally divided into those which directly affect the mitochondrial PT pore and are not energy-dependent (CsA, free Ca²⁺, ADP_ex, and Mg²⁺), and those which affect the energy-dependent calcium phosphate sequestration process (ADP_mt, CATR, local anesthetics). We also conclude that ANT affects PT by changing mitochondrial capacity for energization.
Hypothyroidism renders liver mitochondria resistant to the opening of membrane permeability transition pore
1998, Biochimica et Biophysica Acta - Molecular Basis of Disease
Membrane permeability was examined in liver mitochondria isolated from hypothyroid rats. It was found that such a thyroid status provides substantial protection from membrane leakiness as induced by Ca²⁺ loading. Thus, these mitochondria are less prone to undergoing permeability transition than mitochondria from euthyroid rats. The above conclusion was reached on the basis of the following two facts: (1) hypothyroid mitochondria are not strictly dependent on the addition of ADP to retain high matrix Ca²⁺ concentrations, and (2) carboxyatractyloside, antimycin A or carbonyl cyanide-m-chlorophenyl hydrazone failed to promote Ca²⁺ efflux. We discuss the possible relevance of the low content of membrane cardiolipin as well as the low expression of the adenine nucleotide translocase as responsible for the resistance to membrane damage.

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The nonspecific inner membrane pore of liver mitochondria: Modulation of cyclosporin sensitivity by ADP at carboxyatractyloside-sensitive and insensitive sites

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biol. Chem.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

FEBS Lett.

FEBS Lett.

Biochem. Biophys. Res. Comm.

Am. J. Physiol.