The motion of the sensor regions in a mitochondrial voltage-gated channel called VDAC were probed by attaching biotin at specific locations and determining its ability to bind to added streptavidin. Site-directed mutagenesis was used to introduce single cysteine residues into Neurospora 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 streptavidin addition: in type 1, channel conductance was reduced, but voltage gating could proceed; in type 2, channels were locked in a closed state. The result at type 1 sites is interpreted as streptavidin binding to sites in static regions close to the channel opening. The binding sterically interferes with ion flow. The result at type 2 sites indicates that these are located on a mobile domain and coincide with the previously identified sensor regions. The findings are consistent with closure resulting from the movement of a domain from within the transmembrane regions to the membrane surface. No single site was accessible to streptavidin from both membrane surfaces, indicating that the motion is limited. From the streptavidin-induced reduction in conductance at type 1 sites, structural information was obtained about the location of these sites.

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Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Song J, Midson C, Blachly-Dyson E, Forte M, Colombini M

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