Chemical Modification of Arginines by 2,3-Butanedione and Phenylglyoxal Causes Closure of the Mitochondrial Permeability Transition Pore

Abstract

We have investigated the role of arginine residues in the regulation of the mitochondrial permeability transition pore, a cyclosporin A-sensitive inner membrane channel. Isolated rat liver mitochondria were treated with the arginine-specific chemical reagent 2,3-butanedione or phenylglyoxal, followed by removal of excess free reagent. After this treatment, mitochondria accumulated Ca 2؉ normally, but did not undergo permeability transition following depolarization, a condition that normally triggers opening of the permeability transition pore. Inhibition by 2,3-butanedione and phenylglyoxal correlated with matrix pH, suggesting that the relevant arginine(s) are exposed to the matrix aqueous phase. Inhibition by 2,3-butanedione was potentiated by borate and was reversed upon its removal, whereas inhibition by phenylglyoxal was irreversible. Treatment with 2,3-butanedione or phenylglyoxal after induction of the permeability transition by Ca 2؉ overload resulted in pore closure despite the presence of 0.5 mM Ca 2؉. At concentrations that were fully effective at inhibiting the permeability transition, these arginine reagents (i) had no effect on the isomerase activity of cyclophilin D and (ii) did not affect the rate of ATP translocation and hydrolysis, as measured by the production of a membrane potential upon ATP addition in the presence of rotenone. We conclude that reaction with 2,3-butanedione and phenylglyoxal results in a stable chemical modification of critical arginine residue(s) located on the matrix side of the inner membrane, which, in turn, strongly favors a closed state of the pore.Mammalian mitochondria contain an inner membrane channel, the permeability transition pore (PTP) 1 , that, when fully open, permits free diffusion of solutes with a molecular mass of up to ϳ1500 Da (1, 2). The PTP is controlled by several ligands as well as by the ⌬⌿ across the inner membrane; high values of ⌬⌿ favor the closed state, whereas a decrease in ⌬⌿ may increase the open probability (3). High matrix Ca 2ϩ concentrations, P i , and oxidation of intramitochondrial pyridine nucleotides promote pore opening, whereas ADP, H ϩ , and CsA cause inhibition (1, 2). CsA binds to mitochondrial matrix CyP-D (4), which may cause pore inhibition by releasing CyP-D from its putative binding sites on the pore (5, 6). Based on the effect of atractyloside and bongkrekate on the permeability transition (7) and based on the finding that the ANT may form a Ca 2ϩ -dependent large conductance channel in phospholipid bilayers (8), it has been suggested that the pore may be formed by the ANT (7).The physiological function of the permeability transition is not known, but circumstantial evidence suggests that it is involved in Ca 2ϩ homeostasis (9, 10) and linked to cell death. This notion has gained momentum with the findings that (i) Bax and Bcl-XL , two channel-forming proteins of the Bcl-2 superfamily localized mainly to mitochondria (11,12), may affect the PTP and therefore release of the apoptosis-inducing factor, a mitochondrial caspa...

Inhibition of N-Methyl-d-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential

“…It has been reported that the mitochondrial permeability transition pore can be regulated by the oxidation of sulfhydryl groups and membrane surface potential (61) and following ⌬⌿ m depolarization, would likely favor the formation of a disulfide bridge with C-R(7) within the pore, as supported by studies showing that dithiol, through dithiol-disulfide interconversions, control the pore conformation of the mPTP (62). The anchoring of the positively charged C-R(7) peptide within the channel could then interfere with the otherwise massive calcium entry into the mitochondrial matrix.…”

Modification of Permeability Transition Pore Arginine(s) by Phenylglyoxal Derivatives in Isolated Mitochondria and Mammalian Cells

“…The mechanism for voltage sensing by voltage-dependent ion channels is based on the movement and reorientation of positively charged amino acids in the transmembrane electric field (27). Based on (i) the effect of arginine modification on PTP conformation and (ii) the exquisite sensitivity of PTP opening and closing to small structural differences between the arginine-reactive compounds, we have proposed that the arginine(s) is located on a putative voltage-sensing element of the PTP (19,20). Covalent modification of the voltage-sensing arginines is expected to exert a large influence on sensor orientation and therefore on pore conformation.…”

Antimelanoma Activity of Apoptogenic Carbonyl Scavengers

“…5), an apoptogenic kinase inhibitor and inducer of mitochondrial depolarization, swelling, outer membrane rupture, and cytochrome c release (Scarlett et al, 2000;Tafani et al, 2001;Duan et al, 2003). Previous work has demonstrated that RCS, including MG, glyoxal, and the synthetic cell-permeable MG analog PG, block MPT pore opening, transmembrane potential dissipation, and mitochondrial swelling induced by high Ca 2ϩ and ganglioside GD3, known inducers of mitochondrial pathways of apoptosis by MPT pore opening (Eriksson et al, 1998;Johans et al, 2005). Consistent with involvement of MPT pore opening in carbonyl scavenger induction of melanoma cell apoptosis, cyclosporine A treatment suppressed DMC-induced apoptosis (Fig.…”