CNR - Institute of Neuroscience CNR
Institute of Neuroscience
 

Project

Mitochondrial ion channels

Summary


Electrophysiological and biochemical techniques are used to identify and characterise ion-conducting channels of the mitochondrial inner membrane to improve our understanding of the permeability properties of this energy-transducing membrane. Their possible roles in mitochondrial and cellular physiology are investigated. Background. In order for transmembrane electrochemical ion gradients to exist, the permeability of biological membranes to ions must be carefully regulated. This is especially true of the inner membrane of mitochondria (IMM), the site at which food-derived energy is stored in the form of an electrochemical proton gradient which provides the proximal input for endoergic ATP synthesis. The complex biochemical processes taking place in mitochondria, and the optimisation of organelle functionality require however an intense transmembrane traffic of organic molecules and ions. Along with a large number of carriers, exchangers and "pumps", the inner mitochondrial membrane contains ion-conducting channels which endow it with controlled permeability to small ions. Aspecific permeation pathways may open in response to conditions such as induction of apoptosis, Ca2+ overload, oxidative insults or possibly, when dealing with isolated mitochondria, as an unwanted consequence of the purification. Ours is one of only a few groups in the world to apply patch-clamping to mitochondria (actually swollen mitoplasts).

Several mitochondrial channels have been identified on the basis of their properties. Their molecular identity is known in some cases, and in all such instances the mitochondrial channels appear to represent subpopulations of channels present also elsewhere. The mechanism(s) responsible for dual targeting of these channels is not known. Project. During past studies on the Mitochondrial Permeability Transition Pore (MPTP; a high-conductance Ca2+- and oxidative stress-induced pore) of the IMM of rat liver mitochondria "background" currents were commonly observed, but were difficult to characterise because the channels presumably responsible for them have relatively low conductances and their activity was often masked by the much larger currents carried by the prevalent MPTP. In mitochondria isolated from cultured cells the incidence of MPTP activity is lower. We are therefore using organellar preparations from cultured cells to study IMM channels at the single-channel level, to verify their presence in lines of various origins, and to investigate their role in the cells' life and death.

Methods

Patch-clamp, applied to swollen mitochondria, is coupled to classical biochemical approaches such as Western blotting and to the use of pharmacological agents. The methods of cellular biology are used in functional studies.

Some results

 

We have recently discovered and studied two K+-selective channels in the IMM. One is KV1.3, a member of the Shaker family of voltage-dependent channels, which is present in the mitochondria (as well as in the plasma membrane) of lymphocytes and lymphocyte-derived cell lines (Szabò et al., 2005). This mitochondrial channel turned out to have a role in the apoptosis of these cells (Szabò et al., 2008). Bax, the best-known pro-apoptotic protein of the Bcl2 family, can, after inserting into the outer membrane of mitochondria, interact with the channel vestibule. This interaction involves a crucial lysine residue believed to protrude from the outer membrane into the periplasmic space of mitochondria. The sequence interval containing this conserved lysine is homologous to sequences in peptide toxin inhibitors of KV channels, all of which include a lysine well-known to be crucial for their inhibitory activity. A depiction of the interaction between Bax (in solution) and a K+ channel (KcsA) tetrameric vestibule is presented below. According to our working model, KV1.3 blockage by Bax results in hyperpolarisation of the IMM, production of ROS, and facilitation of cytochrome c detachment and efflux from mitochondria.

 

The second mt K+ channel we have recently studied represents, again, a mitochondrial population of a channel located mostly in the plasma membrane (De Marchi et al., 2009). This is KCa3.1, a Ca2+-activated channel. Its Ca2+ sensitivity and orientation are such that any significant increase of matrix free Ca2+ is expected to lead to an increase of its activity, with consequences for mitochondrial transmembrane potential, volume and perhaps energy conversion efficiency.

Publications

  • De Marchi U, Sassi N, Fioretti B, Catacuzzeno L, Cereghetti GM, Szabò I, Zoratti M (2009) Intermediate conductance Ca2+-activated potassium channel (KCa3.1) in the inner mitochondrial membrane of human colon cancer cells. Cell Calcium 45:509-16.
  • Zoratti M, De Marchi U, Gulbins E, Szabò I (2009) Novel channels of the inner mitochondrial membrane. Biochim. Biophys. Acta 1787:351-63.
  • Szabó I, Bock J, Grassmé H, Soddemann M, Wilker B, Lang F, Zoratti M, Gulbins E (2008) Mitochondrial potassium channel Kv1.3 mediates Bax-induced apoptosis in lymphocytes. Proc. Natl. Acad. Sci. U.S.A. 105:14861-6.
  • De Marchi U, Szabò I, Cereghetti GM, Hoxha P, Craigen WJ, Zoratti M (2008) A maxi-chloride channel in the inner membrane of mammalian mitochondria. Biochim. Biophys. Acta 1777:1438-48.
  • De Marchi U, Basso E, Szabò I, Zoratti M (0) Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore. Mol. Membr. Biol. 23:521-30.
  • Szabò I, Bock J, Jekle A, Soddemann M, Adams C, Lang F, Zoratti M, Gulbins E (2005) A novel potassium channel in lymphocyte mitochondria. J. Biol. Chem. 280:12790-8.

Grants

The project is supported by a grant from the Italian Association for Cancer Research and by Institutional funds

Collaborations

  • Prof. Ildikò Szabò, Dept of Biology of the University of Padova.
  • Prof. Erich Gulbins, University of Essen (Germany).

 

PI photo

Mario Zoratti

Contact information

email  E-mail

email  049 8276054

Participating staff

Francesco Tombola

Umberto De Marchi

Nicola Sassi

Luigi Leanza