Biophysical and electropharmacological properties of single mitoKATP channel in rat brain mitochondrial inner membrane

Introduction: Different ATP-sensitive potassium channels have been detected in the mitochondrial inner membrane of cells. They are suggested to be involved in cell processes including cell protection. Here, we characterized the biophysical and electropharmacological properties of a KATP channel in the brain mitochondrial inner membranes. Methods: After removing and homogenizing the rat brain, the supernatant was separately centrifuged in MSE digitonin, H2O and Na2CO3 and mitochondrial inner membrane vesicles were obtained in MSE solution. L-α- Phosphatidylcholine (membrane lipid) was extracted from fresh egg yolk. Bilayer lipid membranes were formed in a 150 μm diameter hole. All recordings were filtered at 1 kHz and stored at a sampling rate of 10 kHz for offline analysis by PClamp10. Results: Single channel recordings revealed a channel with a slope conductance of 143 ± 7pS in voltage dependent 200 mM KCl cis/50 mM KCl trans. The closed dwell time distributions indicated one and at least two exponential components at positive and negative potentials, respectively. The open dwell time events were fitted to two exponential functions. The block by ATP and glibenclamide characterized this channel as the KATP channel. We also showed that 4- AP and TEA inhibited the channel activities. The activity of channel was not influenced by iberiotoxin, a BK channel blocker, and 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-sensitive potassium channels. Western blotting with antibodies directed against Kir6.1 and SUR2B subunits recognized a blocking-peptide-sensitive with a molecular mass of ~55-64kDa and ~120kDa, respectively. Conclusion: This channel is likely to be involved in maintaining proper homeostasis in brain mitochondria and cell processes.