Electrophysiological characterization of LacY

Abstract

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.

Fig. 1.

Transient currents obtained with wild-type LacY proteoliposomes after a 50 mM sugar concentration jump at t = 1.5 s. The traces in black, red, and green correspond to concentration jumps of lactose (50 mM; ΔLactose), lactulose (50 mM; ΔLactulose), melibiose (50 mM; ΔMelibiose), and sucrose (50 mM; ΔSucrose), respectively. The nonactivating solution (50 mM glucose) and all activating solutions were prepared in 100 mM potassium phosphate at pH 7.6 plus 1 mM DTT. All traces shown were recorded from 1 sensor.

Fig. 2.

Transient currents obtained at 2 different LPR (10 and 5). The solution exchange protocol and the nonactivating solution were as described in Fig. 1, but the activating solution contained 40 mM glucose plus 10 mM lactose. Therefore, the difference between the test and nonactivating solutions represents a 10 mM lactose concentration jump (10 mM ΔLactose). Wild-type LacY proteoliposomes reconstituted at a LPR of 10 (≈1,000 particles per μm²; Fig. S2A) or at a LPR of 5 (≈4,500 particles per μm²; Fig. S2B) were activated with a 10 mM lactose concentration jump. The decay phase of the transient currents is decreased almost 5-fold from a τ_1/2 = 53 ± 2 ms at an LPR of 5 (black trace) to a τ_1/2 = 260 ± 2 ms at an LPR of 10 (gray trace).

Fig. 3.

Effect of pH on the transient currents generated after 50 mM lactose concentration jumps at different pH values. The traces were successively recorded on the same sensor after equilibration was reached and are, therefore, directly comparable. To equilibrate the pH across the proteoliposome membrane after changing the pH of the solutions, the immobilized proteoliposomes are incubated for ≈20 min at the new pH. Subsequent lactose concentration jumps produced constant currents indicating that the pH value had indeed equilibrated. The nonactivating solution contained 50 mM glucose and the activating solutions 50 mM lactose. Both solutions were prepared in 100 mM potassium phosphate buffer at pH 8.5 (black trace), 7.6 (gray trace), or 6.6 (light gray trace) plus 1 mM DTT. (Inset) Dependence of peak currents on lactose concentration at 3 pH values. The nonactivating solution contained 50 mM glucose, the activating solutions a given concentration of x mM lactose plus 50 − x mM glucose to maintain a constant sugar concentration. The solutions were prepared in 100 mM potassium phosphate buffer at a given pH value plus 1 mM DTT, and the pH was equilibrated across the proteoliposome membrane. The peak currents recorded at pH 8.5 for each lactose concentration jump were fitted with a hyperbolic function, and all data obtained with that sensor (every lactose concentration at the 3 pH values) were expressed as fraction of maximum value at pH 8.5 (I_peak^max). This normalization procedure yields datasets that can be directly compared between sensors. For a statistical analysis, the complete dataset was recorded on 3 different sensors, and the averaged values and errors (SE) are shown. From the hyperbolic fits, apparent K_0.5 values with SE were obtained at every pH (Table 1).

Fig. 4.

Transient currents obtained with LacY mutants. The solution exchange protocol and composition of the solutions was the same as described for Fig. 1. The baseline is represented in blue. (A) E325A LacY was reconstituted into liposomes, and activated with 50 mM concentration jumps of lactose (50 mM; ΔLactose), lactulose (50 mM; ΔLactulose), or melibiose (50 mM; ΔMelibiose) at pH 7.6. All traces exhibit virtually identical kinetics and only small differences in magnitude with an exponential decay toward the baseline (τ ≈ 10 ms) followed by a negative phase (τ ≈ 300 ms). (B) Transient currents obtained with C154G LacY proteoliposomes after 50 mM sugar concentration jumps at pH 7.6. The transient currents corresponding to 50 mM ΔLactose or 50 mM ΔLactulose decay mono-exponentially toward the baseline, with time constants of ≈20 ms, whereas the transients observed with 50 mM ΔMelibiose exhibit the largest peak current and a significantly faster exponential decay (τ ≈ 10 ms) followed by a small negative phase.