Homocysteine-Thiolactone Modulates Gating of Mitochondrial Voltage-Dependent Anion Channel (VDAC) and Protects It from Induced Oxidative Stress

Abstract

The gating of the Voltage-Dependent Anion Channel (VDAC) is linked to oxidative stress through increased generation of mitochondrial ROS with increasing mitochondrial membrane potential (ΔΨ_m). It has been already reported that H₂O₂ increases the single-channel conductance of VDAC on a bilayer lipid membrane. On the other hand, homocysteine (Hcy) has been reported to induce mitochondria-mediated cell death. It is argued that the thiol-form of homocysteine, HTL could be the plausible molecule responsible for the alteration in the function of proteins, such as VDAC. It is hypothesized that HTL interacts with VDAC that causes functional abnormalities. An investigation was undertaken to study the interaction of HTL with VDAC under H₂O₂ induced oxidative stress through biophysical and electrophysiological methods. Fluorescence spectroscopic studies indicate that HTL interacts with VDAC, but under induced oxidative stress the effect is prevented partially. Similarly, bilayer electrophysiology studies suggest that HTL shows a reduction in VDAC single-channel conductance, but the effects are partially prevented under an oxidative environment. Gly172 and His181 are predicted through bioinformatics tools to be the most plausible binding residues of HTL in Rat VDAC. The binding of HTL and H₂O₂ with VDAC appears to be cooperative as per our analysis of experimental data in the light of the Hill-Langmuir equation. The binding energies are estimated to be - 4.7 kcal mol^-1 and - 2.8 kcal mol^-1, respectively. The present in vitro studies suggest that when mitochondrial VDAC is under oxidative stress, the effects of amino acid metabolites like HTL are suppressed.

Keywords: Bilayer electrophysiology; Homocysteine-thiolactone (HTL); Hydrogen peroxide (H2O2); Oxidative stress; Voltage-dependent anion channel (VDAC).