Multi-drug
resistance in Gram-negative bacteria is often associated with low permeability of
the outer membrane. To investigate the role of membrane channels in the uptake
of antibiotics, we extract, purify and reconstitute them into artificial planar
membranes. To avoid this time-consuming procedure, here we show a robust
approach using fusion of native outer membrane vesicles (OMV) into planar lipid
bilayer which moreover allows also to some extend the characterization of
membrane protein channels in their native environment. Two major membrane
channels from <i>Escherichia coli</i>, OmpF
and OmpC, were overexpressed from the host and the corresponding OMVs were
collected. Each OMV fusion revealed surprisingly single or only few channel activities.
The asymmetry of the OMV´s translates after fusion into the lipid membrane with
the LPS dominantly present at the side of OMV addition. Compared to
conventional reconstitution methods, the channels fused from OMVs containing
LPS have similar conductance but a much broader distribution. The addition of Enrofloxacin
on the LPS side yields somewhat higher association (<i>k<sub>on</sub></i>) and lower dissociation (<i>k<sub>off</sub></i>) rates compared to LPS-free reconstitution. We
conclude that using outer membrane vesicles is a fast and easy approach for
functional and structural studies of membrane channels in the native membrane.
Within-species variation in OMV cargo proteins: the Myxococcus xanthus OMV pan-proteome