VDAC1-interacting anion transport inhibitors inhibit VDAC1 oligomerization and apoptosis

Mitochondria-mediated apoptosis involves pro-apoptotic protein release from the mitochondria to the cytosol, triggering apoptosis. However, the mechanisms by which apoptotic initiators cross the outer mitochondrial membrane (OMM) remain unclear. The voltage-dependent anion channel 1 (VDAC1), an OMM protein, is central to mitochondria-mediated apoptosis. In previous work, we demonstrated that apoptosis induction is associated with VDAC1 oligomerization, forming a mega-pore that mediates pro-apoptotic protein release. Here, we demonstrated that several known anion transport inhibitors, DIDS, SITS, H₂DIDS, DNDS, and DPC, all interact with VDAC1, as revealed by micro-scale thermophoresis and decreased conductance of bilayer-reconstituted VDAC1. These compounds inhibited apoptosis stimuli-induced release of mitochondrial pro-apoptotic proteins, apoptosis and VDAC1 oligomerization, as monitored by chemical cross-linking or in living cells by BRET2. Moreover, the compounds inhibited VDAC1 oligomerization in isolated mitochondria and as induced by VDAC1 over-expression, suggesting that the inhibitory effect of the tested compounds involved VDAC1.Finally, the compounds also inhibited apoptosis-associated increases in intracellular Ca²⁺, ([Ca²⁺]i), ROS production, mitochondria membrane potential dissipation and the increase in VDAC1 expression levels. The results presented here explored a new mechanism of action for DIDS and its analogs. All inhibited apoptosis via direct interaction with VDAC1 to inhibit its oligomerization and subsequent Cyto c release and apoptosis. Such results may allow the development of a VDAC1-specific inhibitor that would offer substantial insight into the function of VDAC1 in controlling metabolism, energy production, cholesterol transport and apoptosis. Finally, inhibitors of apoptosis could serve in pathological conditions where enhanced apoptosis is found, such as neurodegenerative diseases.