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A Large, Voltage-Dependent Channel, Isolated from Mitochondria by Water-Free Chloroform Extraction

Evgeny Pavlov ^*, Eleonora Zakharian , Christopher Bladen ^*, Catherine T. M. Diao ^*, Chelsey Grimbly ^*, Rosetta N. Reusch  and Robert J. French ^*

^* Department of Physiology and Biophysics, University of Calgary, Alberta, Canada; and Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan, USA

Correspondence: Address reprint requests to Robert J. French, Dept. of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada T2N 4N1. Tel.: 403-220-6893; Fax: 403-283-8731; E-mail: french{at}ucalgary.ca.

We examined ion channels derived from a chloroform extract of isolated, dehydrated rat liver mitochondria. The extraction method was previously used to isolate a channel-forming complex containing poly-3-hydroxybutyrate and calcium polyphosphate from Escherichia coli. This complex is also present in eukaryotic membranes, and is located primarily in mitochondria. Reconstituted channels showed multiple subconductance levels and were voltage-dependent, showing an increased probability of higher conductance states at voltages near zero. In symmetric 150 mM KCl, the maximal conductance of the channel ranged from 350 pS to 750 pS. For voltages >±60 mV, conductance fluctuated in the range of 50–200 pS. In the presence of a 1:3 gradient of KCl, at pH = 7.4, selectivity periodically switched between different states ranging from weakly anion-selective (V_rev –15 mV) to ideally cation-selective (V_rev +29 mV), without a significant change in its conductance. Overall, the diverse, but highly reproducible, channel activity most closely resembled the behavior of the permeability transition pore channel seen in patch-clamp experiments on native mitoplasts. We suggest that the isolated complex may represent the ion-conducting module from the permeability transition pore.

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