Electrophysiological Investigation of the Lactose Permease from Escherichia coli on a Solid-Supported Membrane

Abstract

Electrogenic events associated with the activity of the wild-type lactose permease (LacY) of Escherichia coli were investigated by using proteoliposomes containing purified LacY adsorbed onto a solid-supported membrane. Activation of the proteoliposomes with concentration jumps of different substrates generated transient currents. Analysis of the transient currents at different lipid to protein ratios and different pH values show that the currents represent stationary turnover of LacY. Furthermore, selective inactivation of the substrate binding by alkylation of C148 with N-ethyl maleimide (NEM) suppressed the transient currents, indicating that the transients correspond to the electrogenic activity of LacY.Mutant E325A LacY was used to investigate possible electrogenic steps in the transport cycle unrelated with proton translocation. In addition, electrogenic steps taking place before proton translocation were investigated with C154G LacY, which binds sugar as well as the wild-type but catalyzes very little transport activity. Both mutants show electrogenic activity after activation with different substrates. Therefore, either substrate binding or a conformational change following substrate binding is responsible for these electrical transients. As exchange (but not efflux) is almost voltage independent in wild-type LacY, it is concluded that there are least two electrogenic steps in the transport cycle.