Abstract
Planar bilayers formed from Escherichia coli outer membrane vesicles exhibit conductance properties similar to those previously observed in bilayers reconstituted from aggregates of matrix protein, the major outer membrane protein. Discrete conductance steps are observed, reflecting voltage-dependent transmembrane channels. These exist in clusters which are activated by voltage. After activation, channels close with increasing potentials and reopen reversibly at lower voltage. Depending on the sign of the potential, two distinct closed states of the pores are observed. Cooperative interactions, hysteresis effects, relaxation times, and values of channel conductance depend on cluster size. These properties provide the reference data for the reconstitution of membrane function from individual components. Planar bilayers were formed from vesicles containing either solubilized matrix protein in a homogeneous trimeric state or bacterial glycolipid (lipopolysaccharide), or both. Activation of channel conductance required the presence of glycolipid and the formation of channel clusters, leading to conductance properties of the channels closely resembling those observed in native outer membranes. At very low concentrations of trimers, irreversible association to clusters by lateral diffusion was observed. Nearly quantitative recoveries of channels allowed the assignment of three pores per trimer.