THE SENSOR REGIONS OF VDAC ARE TRANSLOCATED FROM WITHIN THE MEMBRANE TO THE SURFACE DURING THE GATING PROCESSES

The motion of the sensor regions in a mitochondrial voltage-gated chan nel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin. Site-direct ed mutagenesis was used to introduce single cysteine residues into Neu rospora crassa VDAC (naturally lacks cysteine). These were chemically biotinylated and reconstituted into planar phospholipid membranes. In the 19 sites examined, only two types of results were observed upon st reptavidin addition: in type 1, channel conductance was reduced, but v oltage gating could proceed; in type 2, channels were locked in a clos ed state. The result at type 1 sites is interpreted as streptavidin bi nding to sites in static regions close to the channel opening. The bin ding sterically interferes with ion flow. The result at type 2 sites i ndicates that these are located on a mobile domain and coincide with t he previously identified sensor regions. The findings are consistent w ith closure resulting from the movement of a domain from within the tr ansmembrane regions to the membrane surface. No single site was access ible to streptavidin from both membrane surfaces, indicating that the motion is limited. From-the streptavidin-induced reduction in conducta nce at type 1 sites, structural information was obtained about the loc ation of these sites.