Refractive Index Imaging Reveals That Elimination of the ATP Synthase C Subunit Does Not Prevent the Adenine Nucleotide Translocase-Dependent Mitochondrial Permeability Transition.

The mitochondrial permeability transition pore (mPTP) is a large, weakly selective pore that opens in the mitochondrial inner membrane in response to the pathological increase in matrix Ca2+ concentration. mPTP activation has been implicated as a key factor contributing to stress-induced necrotic and apoptotic cell death. The molecular identity of the mPTP is not completely understood. Both ATP synthase and adenine nucleotide translocase (ANT) have been described as important components of the mPTP. Using a refractive index (RI) imaging approach, we recently demonstrated that the removal of either ATP synthase or ANT eliminates the Ca2+-induced mPTP in experiments with intact cells. These results suggest that mPTP formation relies on the interaction between ATP synthase and ANT protein complexes. To gain further insight into this process, we used RI imaging to investigate mPTP properties in cells with a genetically eliminated C subunit of ATP synthase. These cells also lack ATP6, ATP8, 6.8PL subunits and DAPIT but, importantly, have a vestigial ATP synthase complex with assembled F1 and peripheral stalk domains. We found that these cells can still undergo mPTP activation, which can be blocked by the ANT inhibitor bongkrekic acid. These results suggest that ANT can form the pore independently from the C subunit but still requires the presence of other components of ATP synthase.

Investigation of Properties of the Mitochondrial Permeability Transition Pore Using Whole-Mitoplast Patch-Clamp Technique.

The mitochondrial permeability transition pore (mPTP) is a channel in the mitochondrial inner membrane that is activated by excessive calcium uptake. In this study, we used a whole-mitoplast patch-clamp approach to investigate the ionic currents associated with mPTP at the level of the whole single mitochondrion. The whole-mitoplast conductance was at the level of 5 to 7 nS, which is consistent with the presence of three to six single mPTP channels per mitochondrion. We found that mPTP currents are voltage dependent and inactivate at negative potential. The currents were inhibited by cyclosporine A and adenosine diphosphate. When mPTP was induced by oxidative stress, currents were partially blocked by the adenine nucleotide translocase inhibitor bongkrekic acid. Our data suggest that the whole-mitoplast patch-clamp approach is a useful method for investigating the biophysical properties and regulation of the mPTP.

Inorganic Polyphosphate in Mitochondrial Energy Metabolism and Pathology.

In this chapter, the current understanding of the potential roles played by polyphosphate in mitochondrial function with a specific focus on energy metabolism and mitochondrial pathologies caused by stress is summarized. Here we will discuss details of the possible ion transporting mechanisms of mitochondria that might involve polyP and their role in mitochondrial physiology and pathology are discussed.

The very low number of calcium-induced permeability transition pores in the single mitochondrion.

Mitochondrial permeability transition (PT) is a phenomenon of stress-induced increase in nonspecific permeability of the mitochondrial inner membrane that leads to disruption of oxidative phosphorylation and cell death. Quantitative measurement of the membrane permeability increase during PT is critically important for understanding the PT's impact on mitochondrial function. The elementary unit of PT is a PT pore (PTP), a single channel presumably formed by either ATP synthase or adenine nucleotide translocator (ANT). It is not known how many channels are open in a single mitochondrion during PT, which makes it difficult to quantitatively estimate the overall degree of membrane permeability. Here, we used wide-field microscopy to record mitochondrial swelling and quantitatively measure rates of single-mitochondrion volume increase during PT-induced high-amplitude swelling. PT was quantified by calculating the rates of water flux responsible for measured volume changes. The total water flux through the mitochondrial membrane of a single mitochondrion during PT was in the range of (2.5 ± 0.4) × 10-17 kg/s for swelling in 2 mM Ca2+ and (1.1 ± 0.2) × 10-17 kg/s for swelling in 200 µM Ca2+. Under these experimental conditions, a single PTP channel with ionic conductance of 1.5 nS could allow passage of water at the rate of 0.65 × 10-17 kg/s. Thus, we estimate the integral ionic conductance of the whole mitochondrion during PT to be 5.9 ± 0.9 nS for 2 mM concentration of Ca2+ and 2.6 ± 0.4 nS for 200 µM of Ca2+. The number of PTPs per mitochondrion ranged from one to nine. Due to the uncertainties in PTP structure and model parameters, PTP count results may be slightly underestimated. However, taking into account that each mitochondrion has ∼15,000 copies of ATP synthases and ANTs, our data imply that PTP activation is a rare event that occurs only in a small subpopulation of these proteins.

Са2+- and NF-κB-dependent generation of NO in the photosensitized neurons and satellite glial cells.

Photodynamic therapy (PDT) is used for killing of malignant cells in tumors including brain cancer. It can also damage normal neurons and glial cells. Nitric oxide (NO) is known to control PDT-induced cell death. To study the mechanisms that regulate NO generation in photosensitized neurons and glial cells, we used a simple model object - isolated crayfish mechanoreceptor that consists of a single sensory neuron surrounded by glial cells. PDT induced NO generation in glial cells, neuronal dendrites, and, less, in soma and axon. Using modulators of the cytosolic Ca2+ level and nuclear factor-kappa B (NF-κB) activity, we showed that Ca2+ and NF-κB regulate NO generation in the photosensitized neurons and glia. Actually, NO production was stimulated by 4-fold cadmium chloride (CdCl2) concentration in the saline, Ca2+ ionophore ionomycine, or inhibition of Ca2+-ATPase in the endoplasmic reticulum by 2,5-ditert-butylbenzene-1,4-diol (tBuBHQ). Oppositely, CdCl2 or nifedipine, blockers of Ca2+ channels in the plasma membrane, decreased NO generation. NO production was also inhibited by S-methylthiouronium sulfate (SMT), inhibitor of Ca2+-independent inducible NO synthase. SMT also prevented the stimulation of PDT-induced NO generation by NF-κB activator prostratin. This suggests the involvement of both Ca2+-dependent neuronal NO synthase and Ca2+-independent inducible NO synthase, which is regulated by NF-κB, in NO production in the crayfish neurons and glia.

ATP Synthase C-Subunit-Deficient Mitochondria Have a Small Cyclosporine A-Sensitive Channel, but Lack the Permeability Transition Pore.

Permeability transition (PT) is an increase in mitochondrial inner membrane permeability that can lead to a disruption of mitochondrial function and cell death. PT is responsible for tissue damage in stroke and myocardial infarction. It is caused by the opening of a large conductance (∼1.5 nS) channel, the mitochondrial PT pore (mPTP). We directly tested the role of the c-subunit of ATP synthase in mPTP formation by measuring channel activity in c-subunit knockout mitochondria. We found that the classic mPTP conductance was lacking in c-subunit knockout mitochondria, but channels sensitive to the PT inhibitor cyclosporine A could be recorded. These channels had a significantly lower conductance compared with the cyclosporine A-sensitive channels detected in parental cells and were sensitive to the ATP/ADP translocase inhibitor bongkrekic acid. We propose that, in the absence of the c-subunit, mPTP cannot be formed, and a distinct cyclosporine A-sensitive low-conductance channel emerges.