Electrophysiological Characterization of Transport Across Outer Membrane Channels from Gram-Negative Bacteria in Their Native Environment

Multi-drug-resistance in Gram-negative bacteria is often associated with low permeability of outer membrane. To investigate the role of membrane protein channels in the passage of antibiotics, we extract, purify, reconstitute them into artificial bilayer. Here we demonstrate that using a fusion of native outer membrane vesicles (OMV) facilitates channel reconstitution into bilayer and allows to characterize them in their native environment. Proteins from E. coli (OmpF, OmpC) were overexpressed from the host, and the corresponding OMVs were collected. Each OMV fusion revealed surprisingly only a few channel activities. The asymmetry of the OMV translates after fusion into bilayer with the LPS dominantly present at OMV addition side. Compared to conventional methods, channels fused from OMVs have similar conductance but broader distribution. The further addition of Enrofloxacin yielded higher association but lower dissociation rates attribute to the presence of LPS. We conclude using OMV is a robust approach for functional and structural studies of membrane channels in the native membrane.