Electrophysiological behavior of the TolC channel-tunnel in planar lipid bilayers

Escherichia coli TolC assembles into the unique channel-tunnel structure spanning the outer membrane and periplasmic space. The structure is constricted only at the periplasmic entrance of the tunnel and this must be opened to allow export of substrates bound by cognate inner membrane complexes. We have investigated the electrophysiological behavior of TolC reconstituted into planar lipid bilayers, in particular the influence of the membrane potential, the electrolyte concentration and pH. TolC inserted in one orientation into the membrane. The resultant pores were stable and showed no voltage-dependent opening or closing. Nevertheless, TolC could adopt up to three conductance substates. The pores were cation-selective with a permeability ratio of potassium to chloride ions of 16.5. The single-channel conductance was higher when the protein was inserted from the side with negative potential. It showed a nonlinear dependence on the concentration of the electrolyte in the bulk solution and decreased as the pH was lowered. The calculated pK of the apparent closing was 4.5. The electrophysiological characterization is discussed in relation to the TolC structure, in particular the periplasmic entrance.