Mitochondrial voltage-dependent anion channel is involved in dopamine-induced apoptosis

Neuronal NMB cells were used to determine changes in gene expression upon treatment with dopamine. Twelve differentially expressed cDNAs were identified and cloned, one of them having 99.4% sequence homology with isoform 2 of a voltage-dependent anion channel (VDAC-2). The known role of VDAC, a mitochondrial outer-membrane protein, in transport of anions, pore formation, and release of cytochrome C prompted us to investigate the possible role of VDAC gene family in dopamine-induced apoptosis. Semi-quantitative PCR analysis indicated that expression of the three VDAC isoforms was reduced by dopamine. Immunoblotting with anti-VDAC antibodies detected two VDAC protein bands of 33 and 34 kDa. Dopamine decreased differentially the immunoreactivity of the 34 kDa protein. Whether the decrease in VDAC expression influence the mitochondrial membrane potential (DeltaPsim) was determined with the dye Rhodamine-123. Dopamine indeed decreased the mitochondrial DeltaPsim, but the maximum effect was observed within 3 h, prior to the decrease in VDAC mRNA or protein levels. Cyclosporin A, a blocker of the mitochondrial pore complex, prevented the decrease in DeltaPsim, but did not rescue the cells from dopamine toxicity. To elucidate possible involvement of protease caspases in dopamine-induced apoptosis, the effect of the caspase inhibitor z-Val-Ala-Asp(Ome)-FMK (zVAD) was determined. zVAD decreased dopamine toxicity, yet it did not rescue the mitochondrial DeltaPsim drop. Dopamine also decreased ATP levels. Finally, transfection of NMB cells with pcDNA-VDAC decreased the cytotoxic effect of dopamine. These findings are in agreement with the notion that the mitochondria, and VDAC, are important participants in dopamine-induced apoptosis.