Evidences for a new cation channel in the brain mitochondrial inner membrane

Introduction: Previous studies and our works have indicated several cation channels in the rat brain mitochondrial inner membrane. In this work, we report the single-channel characterization of a cation channel from the rat brain mitochondrial inner membrane incorporated into a planar lipid bilayer. Methods: After removing and homogenizing the adult rat brain, its supernatant was centrifuged in MSE-digitonin, H2O and Na2CO3, separately. Then, mitochondrial inner membrane vesicles were prepared in MSE solution. L-α- Phosphatidylcholine (for preparing membrane lipid bilayer) was extracted from fresh egg yolk. Bilayer lipid membranes were formed in a 150 μm diameter hole. All single channel recordings were filtered at 1 kHz and stored at a sampling rate of 10 kHz for offline analysis by PClamp10. Statistical analysis was performed based on Markov noise free single channel analysis. Results: The channel conductance was 93 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared voltage-independent at -50 to +40 mV. The main characteristic of channel gating was its marked bursting behavior. Adding 10 mM 4-aminopyridine (4-AP) at positive and negative potentials inhibited the channel activities. Conclusion: These results demonstrate that a new cation channel, present in the brain mitochondrial inner membrane, displays different kinetics and biophysical properties than those classically described for brain mitochondria.