Electrophysiological characterization of LacY

Electrogenic events due to the activity of wild-type lactose permease from Escherichia coli (LacY) were investigated with proteoliposomes containing purified LacY adsorbed on a solid-supported membrane electrode. Downhill sugar/H+ symport into the proteoliposomes generates transient currents. Studies at different lipid-to-protein ratios and at different pH values, as well as inactivation by N-ethylmaleimide, show that the currents are due specifically to the activity of LacY. From analysis of the currents under different conditions and comparison with biochemical data, it is suggested that the predominant electrogenic event in downhill sugar/H+ symport is H+ release. In contrast, LacY mutants Glu-325 -> Ala and Cys-154 -> Gly, which bind ligand normally, but are severely defective with respect to lactose/H+ symport, exhibit only a small electrogenic event on addition of LacY-specific substrates, representing 6% of the total charge displacement of the wild-type. This activity is due either to substrate binding per se or to a conformational transition after substrate binding, and is not due to sugar/H+ symport. We propose that turnover of LacY involves at least 2 electrogenic reactions: (i) a minor electrogenic step that occurs on sugar binding and is due to a conformational transition in LacY; and (ii) a major electrogenic step probably due to cytoplasmic release of H+ during downhill sugar/H+ symport, which is the limiting step for this mode of transport.