Voltage dependent anion channel and its interaction with N-acetyl-L-Cysteine (NAC) under oxidative stress on planar lipid bilayer

Abstract

Mitochondria are the major source of Hydrogen Peroxide (H₂O₂), a reactive oxygen species, in the cells. The reactive oxygen species generated by the mitochondria oxidize major proteins including Voltage Dependent Anion Channel (VDAC). We were interested to know how the effect of H₂O₂ is countered by antioxidants present around the mitochondria. N-Acetyl-l-Cysteine (NAC) is a naturally existing antioxidant in the cells. Keeping this in view, the modulatory effect of antioxidant NAC on H₂O₂ oxidized VDAC has been investigated through in vitro electrophysiological studies. First, the effect of H₂O₂ and NAC was studied on independently incorporated single-channel VDAC. It was observed that NAC suppresses VDAC conductance with a half-maximal inhibitory concentration (IC₅₀) of ∼1.04 μM. In contrast, H₂O₂ enhances VDAC conductance. Later, oxidative stress was induced by H₂O₂ on VDAC increased conductance with half-maximal effective concentration (EC₅₀) of ∼302 nM. An application of 1 μM NAC on H₂O₂ treated (300 nM) VDAC reversed the effect of oxidation. In the next step, NAC and H₂O₂ were added in reverse order. When oxidative stress was induced using H₂O₂, reduction in conductance by NAC was 4.5 ± 0.404 nS. The change in conductance is nearly 6.3%. However, if antioxidant NAC was incubated first followed by H₂O₂ treatment, the conductance of VDAC was 3.09 ± 0.27 nS. The change in conductance is near 33%. Both H₂O₂ and NAC also affected various conducting states of VDAC. In-silico studies indicated the binding of NAC at Lysine and Glutamic acid of VDAC. Hence, NAC was found to be effective in protection of VDAC against H₂O₂-induced oxidative stress due to its strong binding.

Keywords: Antioxidant; Bilayer electrophysiology; Docking; N-Acetyl-l-cysteine; Voltage-dependent anion channel.