Abstract
Mitochondrial permeability transition is a process marked by a significant increase in the permeability of the inner mitochondrial membrane that can be initiated by massive Ca2+ influx into mitochondria with or without oxidative stress. The increase in membrane permeability is due to induction of a gigantic proteinaceous pore in the inner mitochondrial membrane called the permeability transition pore (PTP). The detrimental role of PTP induction in neuronal mitochondria in various neuropathologies is well documented. There are different methods to study PTP in isolated mitochondria and in live cells. In the present chapter, we describe a method to monitor PTP induction in individual isolated brain mitochondria. Although this method is not often used, it could be an excellent addition to the arsenal of methodologies utilized in studies of PTP. In addition, we present here detailed description of a method used by us to isolate rat or mouse brain nonsynaptic and synaptic mitochondria.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bernardi P, Krauskopf A, Basso E et al (2006) The mitochondrial permeability transition from in vitro artifact to disease target. FEBS J 273:2077–2099
Bernardi P, Rasola A (2007) Calcium and cell death: the mitochondrial connection. Subcell Biochem 45:481–506
Bernardi P (1999) Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79:1127–1155
Brustovetsky N, Brustovetsky T, Jemmerson R et al (2002) Calcium-induced cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane. J Neurochem 80:207–218
Huser J, Rechenmacher CE, Blatter LA (1998) Imaging the permeability pore transition in single mitochondria. Biophys J 74:2129–2137
Vergun O, Votyakova TV, Reynolds IJ (2003) Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria. Biophys J 85:3358–3366
Vergun O, Reynolds IJ (2004) Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+. Biophys J 87:3585–3593
Vergun O, Reynolds IJ (2005) Distinct characteristics of Ca(2+)-induced depolarization of isolated brain and liver mitochondria. Biochim Biophys Acta 1709:127–137
Shalbuyeva N, Brustovetsky T, Brustovetsky N (2007) Lithium desensitizes brain mitochondria to calcium, antagonizes permeability transition, and diminishes cytochrome C release. J Biol Chem 282:18057–18068
Brustovetsky T, Brittain MK, Sheets PL et al (2011) KB-R7943, an inhibitor of the reverse Na+/Ca2+ exchanger, blocks N-methyl-D-aspartate receptor and inhibits mitochondrial complex I. Br J Pharmacol 162:255–270
Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Percoll density gradient centrifugation. J Neurochem 55:698–707
Wojtczak L, Wojtczak AB, Ernster L (1969) The inhibition of succinate dehydrogenase by oxalacetate. Biochim Biophys Acta 191:10–21
Brown MR, Sullivan PG, Dorenbos KA et al (2004) Nitrogen disruption of synaptoneurosomes: an alternative method to isolate brain mitochondria. J Neurosci Methods 137:299–303
Acknowledgment
The authors are grateful to Jessica Pellman and James Hamilton (Department of Pharmacology and Toxicology, Indiana University School of Medicine) for help with preparing Fig. 2. This work was supported by Indiana Spinal Cord and Brain Injury Research Fund grant and NIH/NINDS grant R01 NS078008 to N.B.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Brustovetsky, N., Brustovetsky, T. (2017). Monitoring of Permeability Transition Pore Openings in Isolated Individual Brain Mitochondria. In: Strack, S., Usachev, Y. (eds) Techniques to Investigate Mitochondrial Function in Neurons. Neuromethods, vol 123. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6890-9_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6890-9_10
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6888-6
Online ISBN: 978-1-4939-6890-9
eBook Packages: Springer Protocols