VDAC1 inhibitor DIDS uncouples respiration in isolated tissue mitochondria and induces mitochondrial hyperfusion in mammalian cells.

Mitochondrial outer membrane protein, voltage-dependent anion channel 1 (VDAC1), is a gatekeeper of transport, metabolism, and cellular apoptosis. Ablation of VDAC1 or treatment with small molecular VDAC1 inhibitors often causes metabolic reprogramming in cells. However, the mechanism of VDAC1-mediated reprogramming of mitochondrial oxidative phosphorylation (OXPHOS) is still unclear. To address this problem, we tested how the high-affinity VDAC1 inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), changes cell viability and mitochondrial functions. The IC50 value of DIDS was found 508 µM and 580 µM after 24 h of treatment on human osteosarcoma U2OS and mouse NIH-3T3 fibroblast cells. Moreover, when we inhibited mitochondrial OXPHOS by oligomycin A, 500 µM DIDS was found to uncouple the respiration like the conventional uncoupler CCCP in both the cells. Additionally, we observed that 50-200 µM DIDS, even after 2 h of treatment, depolarizes mitochondrial membrane potential. Also, brief DIDS treatment leads to an increase in cell population with hyperfused mitochondria and attenuation of DRP1 recruitment to mitochondria in U2OS cells. However, no significant alteration in the steady-state level of mitochondrial respiratory chain complex I and complex V subunits was noticed after DIDS treatment. Similar to cell lines, DIDS treatment also showed significant respiratory uncoupling in isolated mitochondria prepared from the normal muscle, liver, and sarcoma tumor tissues of mice. Finally, in silico modeling using AutoDock Vina and AlphaFold3 identified that DIDS binds inside the beta-barrel structure of VDAC1. Together, our findings directly demonstrate that DIDS binds to the VDAC1 inner pocket, uncouples OXPHOS, and promotes mitochondrial hyperfusion.