The roles of phosphate and the phosphate carrier in the mitochondrial permeability transition pore - PubMed (original) (raw)

The roles of phosphate and the phosphate carrier in the mitochondrial permeability transition pore

Pinadda Varanyuwatana et al. Mitochondrion. 2012 Jan.

Abstract

Phosphate activation of the mitochondrial permeability transition pore (MPTP) opening is well-documented and could involve the phosphate carrier (PiC) that we have proposed is the pore's cyclophilin-D binding component. However, others have reported that following CyP-D ablation Pi inhibits MPTP opening while cyclosporine-A (CsA) inhibits MPTP opening only when Pi is present. Here we demonstrate that Pi activates MPTP opening under all energised and de-energised conditions tested while CsA inhibits pore opening whether or not Pi is present. Using siRNA in HeLa cells we could reduce PiC expression by 65-80% but this inhibited neither mitochondrial calcium accumulation nor MPTP opening.

Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

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Figures

Fig. 1

Fig. 1

CsA-sensitive MPTP opening does not require phosphate. In panel A energised liver mitochondria were incubated in KCl medium containing 20 mM Pi or Asi with or without 1 μM CsA as indicated. Fura-6F and Rhodamine 123 fluorescence were continuously monitored together with light scattering and additions of CaCl2 made as indicated. In panel B mitochondria were incubated in de-energised KCl medium supplemented with 1 mM Pi or Asi with or without 1 μM CsA as indicated, and MPTP opening was determined following addition of 50 μM (free) Ca2+ by monitoring swelling (decrease in A520). Panel C shows the PEG-induced shrinkage of pre-swollen mitochondria incubated in KCl medium containing 60 μM free [Ca2+] in the presence or absence of 1 mM Pi or Asi with or without 1 μM CsA as indicated. In panel D mean data (± S.E.M.; n = 3) are presented for the sensitivity of MPTP opening to [Ca2+] determined by the PEG-shrinkage assay in KCl medium supplemented with either or both 5 mM Pi and 1 μM CsA as indicated. In order to normalise data between different batches of mitochondria rates of shrinking are expressed as a percentage of the rate in the presence of 60 μM Ca2+ and no phosphate. In panel E mean data (± S.E.M.; n = 3) are presented for the sensitivity of MPTP opening to Pi or Asi determined by the PEG-shrinkage assay in KCl medium at 60 μM [Ca2+]free. Further details of all methods used are given under “Materials and methods” (Section 2). Control (Con) refers to KCl medium in the absence of Pi or Asi.

Fig. 2

Fig. 2

MPTP opening in mitochondria of HeLa cell subject to PiC knockdown. After 72 h of transfection with scrambled (panel A) or PiC siRNA (panel B), cells were harvested, washed and permeabilised with digitonin for measurement of MPTP opening. This was performed under energised conditions using Fura-6F and Rhodamine 123 fluorescence together with light scattering. Additions of CaCl2 were made as indicated. Panel C shows the expression of the PiC in cells transfects with scramble (Scr) or PiC siRNA as determined by Western blotting. CyP-D was used as a mitochondrial marker to confirm equivalent loading. Scanning the film revealed that the PiC knockdown was about 65%. Further details of all methods used are given under “Materials and methods” (Section 2).

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