Characterization of biophysical properties of single chloride channel in rat brain mitochondrial inner membrane by channel incorporation into bilayer lipid membrane

Introduction: Recent studies have shown the presence of Cl channels in heart and liver mitochondrial membranes. In this work, we have characterized the functional profile of a Cl channel from rat brain mitochondria. Methods: After removing and homogenizing the rat brain, the supernatant was separately centrifuged in MSEdigitonin, H2O and Na2CO3 and mitochondrial inner membrane vesicles were obtained in MSE solution. L-αPhosphatidylcholine (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. Statistical analysis was performed based on Markov noise free single channel analysis. Results: Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that the mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into the planar lipid bilayers revealed an anion selective channel with a conductance of 301 pS in 200 mM KCl cis/50 mM KCl trans. The channel open probability appeared to be voltage dependent and the channel was active between the voltages of -40 and +20 mV. Adding 10 μM DIDS to the side corresponding to the cell internal medium caused a strong inhibition of the channel activity. Conclusion: This channel is likely to be involved in maintaining proper pH, membrane potential, ATP synthesis, and cell protection.