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Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1.

Crichton PG, Parker N, Vidal-Puig AJ, Brand MD - Biosci. Rep. (2009)

Bottom Line: Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation.When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1).We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

View Article: PubMed Central - PubMed

Affiliation: MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK. pgc@mrc-mbu.cam.ac.uk

ABSTRACT
The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

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Related in: MedlinePlus

Calcium- and erythritol-induced swelling of BAT mitochondria from wild-type and Ucp1KO miceInverse of A520 (A−1) associated with the swelling of wild-type mitochondria (solid lines) and Ucp1KO mitochondria (dashed lines). (A) Swelling induced by 500 μM free Ca2+ (a, b). No calcium controls (c, d). (B) Spontaneous swelling induced by addition of mitochondria to erythritol (a, b) or mannitol medium (c, d). Typical traces are shown and are representative of three repeated experiments.
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Figure 2: Calcium- and erythritol-induced swelling of BAT mitochondria from wild-type and Ucp1KO miceInverse of A520 (A−1) associated with the swelling of wild-type mitochondria (solid lines) and Ucp1KO mitochondria (dashed lines). (A) Swelling induced by 500 μM free Ca2+ (a, b). No calcium controls (c, d). (B) Spontaneous swelling induced by addition of mitochondria to erythritol (a, b) or mannitol medium (c, d). Typical traces are shown and are representative of three repeated experiments.

Mentions: An initial assessment of mPTP formation in BAT mitochondria from Ucp1KO mice suggested that the rates of Ca2+-induced swelling were lower than in mitochondria from wild-type mice (traces b and a respectively, Figure 2A). Caution must be taken, however, as these differences could have been due to general differences in the light scattering properties of Ucp1KO and wild-type mitochondria that were unrelated to mPTP. Indeed, a preliminary assessment of the dependence of A−1 on osmolality−1 for Ucp1KO mitochondria indicated a linear, but shallower, relationship compared with wild-type mitochondria (results not shown). This suggests that the two mitochondrial populations were morphologically different and potentially exhibited different increments in A−1 for a specific amount of solute imported during permeability measurements.


Not all mitochondrial carrier proteins support permeability transition pore formation: no involvement of uncoupling protein 1.

Crichton PG, Parker N, Vidal-Puig AJ, Brand MD - Biosci. Rep. (2009)

Calcium- and erythritol-induced swelling of BAT mitochondria from wild-type and Ucp1KO miceInverse of A520 (A−1) associated with the swelling of wild-type mitochondria (solid lines) and Ucp1KO mitochondria (dashed lines). (A) Swelling induced by 500 μM free Ca2+ (a, b). No calcium controls (c, d). (B) Spontaneous swelling induced by addition of mitochondria to erythritol (a, b) or mannitol medium (c, d). Typical traces are shown and are representative of three repeated experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805926&req=5

Figure 2: Calcium- and erythritol-induced swelling of BAT mitochondria from wild-type and Ucp1KO miceInverse of A520 (A−1) associated with the swelling of wild-type mitochondria (solid lines) and Ucp1KO mitochondria (dashed lines). (A) Swelling induced by 500 μM free Ca2+ (a, b). No calcium controls (c, d). (B) Spontaneous swelling induced by addition of mitochondria to erythritol (a, b) or mannitol medium (c, d). Typical traces are shown and are representative of three repeated experiments.
Mentions: An initial assessment of mPTP formation in BAT mitochondria from Ucp1KO mice suggested that the rates of Ca2+-induced swelling were lower than in mitochondria from wild-type mice (traces b and a respectively, Figure 2A). Caution must be taken, however, as these differences could have been due to general differences in the light scattering properties of Ucp1KO and wild-type mitochondria that were unrelated to mPTP. Indeed, a preliminary assessment of the dependence of A−1 on osmolality−1 for Ucp1KO mitochondria indicated a linear, but shallower, relationship compared with wild-type mitochondria (results not shown). This suggests that the two mitochondrial populations were morphologically different and potentially exhibited different increments in A−1 for a specific amount of solute imported during permeability measurements.

Bottom Line: Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation.When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1).We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

View Article: PubMed Central - PubMed

Affiliation: MRC Mitochondrial Biology Unit, Hills Road, Cambridge CB2 0XY, UK. pgc@mrc-mbu.cam.ac.uk

ABSTRACT
The mPTP (mitochondrial permeability transition pore) is a non-specific channel that is formed in the mitochondrial inner membrane in response to several stimuli, including elevated levels of matrix calcium. The pore is proposed to be composed of the ANT (adenine nucleotide translocase), voltage-dependent anion channel and cyclophilin D. Knockout studies, however, have demonstrated that ANT is not essential for permeability transition, which has led to the proposal that other members of the mitochondrial carrier protein family may be able to play a similar function to ANT in pore formation. To investigate this possibility, we have studied the permeability transition properties of BAT (brown adipose tissue) mitochondria in which levels of the mitochondrial carrier protein, UCP1 (uncoupling protein 1), can exceed those of ANT. Using an improved spectroscopic assay, we have quantified mPTP formation in de-energized mitochondria from wild-type and Ucp1KO (Ucp1-knockout) mice and assessed the dependence of pore formation on UCP1. When correctly normalized for differences in mitochondrial morphology, we find that calcium-induced mPTP activity is the same in both types of mitochondria, with similar sensitivity to GDP (approximately 50% inhibited), although the portion sensitive to cyclosporin A is higher in mitochondria lacking UCP1 (approximately 80% inhibited, compared with approximately 60% in mitochondria containing UCP1). We conclude that UCP1 is not a component of the cyclosporin A-sensitive mPTP in BAT and that playing a role in mPTP formation is not a general characteristic of the mitochondrial carrier protein family but is, more likely, restricted to specific members including ANT.

Show MeSH
Related in: MedlinePlus