The voltage-dependent anion-selective channel (VDAC) is a pore-forming protein abundant in the outer mitochondrial membrane. Its presence in extramitochondrial localizations remains controversial, although recently a plasma membrane localization has received support from the identification of a membrane leader sequence in VDAC (Buettner et al. 2000). The biophysical properties of VDAC resembled those of Maxi Cl^– channels found in the plasma membrane of a wide variety of cell types. Maxi Cl^– channels have typically been observed only in excised patches, suggesting their regulation by intracellular signals (Diaz et al. 2001). We have also observed that Maxi Cl^– channels could be reversibly activated by antioestrogens under whole-cell and cell-attached recording conditions (Hardy & Valverde, 1994; Diaz et al. 2001). However, the physiological relevance of these channels remains unknown.

In the present report we have identified in C1300 mouse neuroblastoma cells a plasma membrane isoform of VDAC (pl-VDAC) by RT-PCR using primers directed against the plasma membrane leader sequence of VDAC (Buettner et al. 2000). The use of a monoclonal antibody against the N-terminus of the mitochondrial VDAC (Calbiochem) allowed us to identify a band of ~31 kDa (expected size for VDAC) in C1300 membrane fractions and localized VDAC to the plasma membrane by immunofluorescence confocal microscopy. Moreover, VDAC co-localised with markers of membrane lipid rafts (cholera toxin β subunit) but not caveolin-1.

The relationship between VDAC and Maxi Cl^– currents activated by antioestrogens was investigated using plVDAC antisense oligonucleotides. Transient transfection of C1300 cells with an antisense directed against the membrane leader sequence of VDAC markedly reduced both the expression of VDAC and the Maxi Cl^– current activated by the antioestrogen toremifene. Whole-cell toremifene-activated Maxi Cl^– current density recorded in control untreated cells (195 ± 50 pA pF^-1), β-globin antisense-transfected cells was 220 ± 50 pA pF^-1 (mean ± S.E.M.; n = 7) and 50 ± 13 pA pF^-1 (n = 11) in pl-VDAC antisense transfected cells (P = 0.05, one-way ANOVA followed by Bonferroni post test).

In summary, our data provides strong support for the presence of VDAC protein in the plasma membrane, including specialized micro-domains such as lipid rafts, and suggests that VDAC is the molecular counterpart of the antioestrogen-activated Maxi Cl^– channel of C1300 neuroblastoma cells.

This work was funded by the Human Frontiers Science Program.