The Mitochondrial Permeability Transition Pore-Current Knowledge of Its Structure, Function, and Regulation, and Optimized Methods for Evaluating Its Functional State

Abstract

The mitochondrial permeability transition pore (MPTP) is a calcium-dependent, ion non-selective membrane pore with a wide range of functions. Although the MPTP has been studied for more than 50 years, its molecular structure remains unclear. Short-term (reversible) opening of the MPTP protects cells from oxidative damage and enables the efflux of Ca²⁺ ions from the mitochondrial matrix and cell signaling. However, long-term (irreversible) opening induces processes leading to cell death. Ca²⁺ ions, reactive oxygen species, and changes in mitochondrial membrane potential regulate pore opening. The sensitivity of the pore to Ca²⁺ ions changes as an organism ages, and MPTP opening plays a key role in the pathogenesis of many diseases. Most studies of the MPTP have focused on elucidating its molecular structure. However, understanding the mechanisms that will inhibit the MPTP may improve the treatment of diseases associated with its opening. To evaluate the functional state of the MPTP and its inhibitors, it is therefore necessary to use appropriate methods that provide reproducible results across laboratories. This review summarizes our current knowledge of the function and regulation of the MPTP. The latter part of the review introduces two optimized methods for evaluating the functional state of the pore under standardized conditions.

Keywords: calcium retention capacity; calcium signaling; calcium-induced swelling; mitochondria; mitochondrial permeability transition; mitochondrial permeability transition pore.

Figure 1

One of the latest structural models of the MPTP (ATP synthasome) and important factors regulating its functional state. The MPTP is a supramolecular complex composed of ATP synthase, a phosphate carrier, and an adenine nucleotide translocator, which is in close contact with the respiratory chain. ADP—adenosine diphosphate; ANT—adenine nucleotide translocator; ATP—adenosine triphosphate; BPR—benzodiazepine receptor (translocator protein); CsA—cyclosporin A; Cyp-D—cyclophilin D; EGTA—ethylene glycol tetraacetic acid; HK—hexokinase II; IMM—inner mitochondrial membrane; MCU—mitochondrial calcium uniporter; OMM—outer mitochondrial membrane; Pi—inorganic phosphate; PiC—inorganic phosphate carrier; Roman numerals I, II, III, and IV represent individual respiratory chain complexes; ROS—reactive oxygen species; VDAC—voltage-dependent anion channel. Green arrows indicate factors that activate MPTP opening. Red markers are for agents inhibiting MPTP opening. Modified from [34,38,39].

Figure 2

Changes in the arrangement of mitochondrial membranes induced by the opening of a Ca²⁺-dependent MPTP with subsequent osmotic processes. Mitochondrial swelling is dependent not only on the concentration of Ca²⁺ ions, but also on the time during which calcium ions act on the mitochondria. The abbreviations are defined in Figure 1. Modified from [51,52].

Figure 3

Mitochondrial swelling induced by calcium ions—classic curves (A) and curves obtained after derivation of primary data (B). The recordings show the effect of Ca²⁺ ion concentration on the extent and rate of changes in mitochondrial swelling. Isolated liver mitochondria (0.2 mg protein/mL) were incubated in swelling medium (125 mM sucrose, 65 mM KCl, and 10 mM HEPES at pH 7.2) supplemented with 0.1 mM inorganic phosphate and 10 mM succinate. Swelling was induced by adding calcium chloride between 50–60 s to the final concentration shown in the figure. Mitochondrial swelling was estimated from a decrease in the absorbance of the mitochondrial suspension at 520 nm using a Shimadzu UV 160 spectrophotometer. From these records, it is possible to obtain precise numerical values for a detailed analysis of the effect of MPTP regulatory factors. An evaluation of the extent and rate is discussed in detail in the text and in our original publications [69,70].

Figure 4

Calcium retention capacity of liver mitochondria. The figure shows representative curves of the combined effect of Ca²⁺ ions and inorganic phosphate on the mitochondrial calcium retention capacity. The mitochondrial calcium retention capacity was evaluated using the membrane-impermeable fluorescent probe calcium green-5N on an AMINCO-Bowman Series 2 spectrofluorometer (λex 506 nm, λem 592 nm). Calculation of the calcium retention capacity of isolated mitochondria was performed by multiplying the amount of added calcium chloride and the number of Ca²⁺ additions, related to mitochondrial protein concentration. Isolated liver mitochondria (0.4 mg protein/mL) were incubated in swelling medium supplemented with 10 mM succinate, 1 μM calcium green-5N, and inorganic phosphate and cyclosporine A, as shown in the figure. The amount of one Ca²⁺ addition was 1.25 nmol (in 1 mL volume). Only MPTP opening in the presence of CsA was induced by high amounts of added calcium (8 × 1.25 nmol Ca²⁺; the last two additions were 10 and 30 nmol Ca²⁺, respectively). An evaluation and calculation of calcium retention capacity is discussed in detail in the text and in our original publications [68,71].