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Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria
13:42

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

Published on: September 7, 2012

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition

J G Pastorino1, M Tafani, R J Rothman

  • 1Department of Pathology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

The Journal of Biological Chemistry
|October 26, 1999
PubMed

Insights

Recombinant Bax protein directly opens the mitochondrial permeability transition pore (PTP). Bax induces cytochrome c release and mitochondrial dysfunction, key events in apoptosis, which can be modulated by calcium and other signaling molecules.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Background:

  • Bax overexpression induces cell death via mitochondrial permeability transition (MPT).
  • The precise mechanism of Bax-induced MPT and its regulation requires further elucidation.

Purpose of the Study:

  • To investigate the direct effects of purified recombinant Bax on the mitochondrial permeability transition pore (PTP).
  • To determine how Bax concentration influences PTP opening and mitochondrial function.
  • To explore the role of calcium, ceramide, and GD3 ganglioside in Bax-mediated MPT.

Main Methods:

  • Purification and recombinant expression of Bax protein.
  • Treatment of isolated mitochondria with varying Bax concentrations.
  • Assays for intermembrane protein release (cytochrome c, adenylate kinase), matrix content release (calcein), mitochondrial swelling, and membrane potential.
  • Inhibition studies using cyclosporin A (CSA).
  • Assessment of calcium, ceramide, and GD3 ganglioside effects on Bax-induced MPT.

Main Results:

  • Recombinant Bax directly opened the PTP in a concentration-dependent manner.
  • Low Bax concentrations (125 nM) caused transient PTP opening and release of intermembrane proteins, prevented by CSA.
  • Higher Bax concentrations (250 nM–1 µM) induced sustained PTP opening, mitochondrial swelling, and deenergization (MPT), also prevented by CSA.
  • Calcium, ceramide, and GD3 ganglioside potentiated Bax-induced MPT.
  • Bax-mediated PTP opening resulted in cytochrome c release.

Conclusions:

  • Bax directly mediates the opening of the mitochondrial PTP.
  • Bax-induced PTP opening is a critical step in the release of cytochrome c and subsequent cell death.
  • The MPT induced by Bax can be modulated by intracellular factors like calcium and specific signaling lipids.

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