Jm. Song et al., THE SENSOR REGIONS OF VDAC ARE TRANSLOCATED FROM WITHIN THE MEMBRANE TO THE SURFACE DURING THE GATING PROCESSES, Biophysical journal, 74(6), 1998, pp. 2926-2944
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.