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Biophys J, June 1998, p. 2926-2944, Vol. 74, No. 6
*Department of Biology, University of Maryland, College Park, Maryland 20742, and #Vollum Institute, Oregon Health Science University, Portland, Oregon 97201 USA
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.
Biophys J, June 1998, p. 2926-2944, Vol. 74, No. 6
© 1998 by the Biophysical Society 0006-3495/98/06/2926/19 $2.00
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