6116 Biochemistry, Vol. 49, No. 29, 2010 Garcia-Celma et al. FIGURE 1: Transient currents measured with wild-type LacY pro- teoliposomes after 100 mM lactose (A) or melibiose (B) concentra- tion jumps at different pH values as indicated. Nonactivating solutions contained 100 mM glucose while activating solutions 100 mM lactose (A) or melibiose (B). All solutions where prepared in 100 mM potassium phosphate buffer at the indicated pH with 1 mM DTT. All traces were recorded at the same sensor. t = 0 corresponds to the time at which the activating solution reaches the sensor surface. was chosen to apply the different solutions (22) at a flow rate of 0.46 mL/s. The nonactivating solution always contained 100 mM glucose, and the activating solution contained lactose or meli- biose at a concentration of 100 mM, unless stated otherwise (Figure 2). All solutions were buffered in 100 mM potassium phosphate buffer at a given pH value with 1 mM dithiothreitol (DTT). Currents were recorded throughout the entire time using a current amplifier set to a gain of 109-108 V/A and a rise time (10-90%) of 3 ms. RESULTS Reduced Downhill Sugar/Hþ Symport by Wild-Type LacY at Low pH. Proteoliposomes reconstituted with purified wild-type LacY were immobilized on an SSM-coated gold elec- trode (the sensor) as described previously (17). The transient currents generated after sugar concentration jumps were re- corded at an improved time resolution [8 ms as compared to 15 ms (17)], and the effect of pH was investigated over a wide range from pH 5.2 to 8.5 (Figure 1A,B). Because all traces were recorded on the same sensor, the amplitudes are directly compar- able. To equilibrate the pH across the proteoliposome membrane after the pH of the solutions had been changed, the immobilized proteoliposomes were incubated for several minutes at the new pH. Subsequent substrate concentration jumps produced FIGURE 2: (A) Transient currents measured for wild-type LacY proteoliposomes after different lactose concentration jumps as in- dicated at pH 5.2. Nonactivating solutions contained 100 mM glucose and activating solutions a concentration of x mM lactose with 100 - x mM glucose to maintain a constant sugar concentration. Solutions were prepared in 100 mM potassium phosphate buffer (pH 5.2) with 1 mM DTT. All traces were recorded at the same sensor. t = 0 corresponds to the time at which the activating solution reaches the sensor surface. The transfer function of the experiment representing the time resolution is shown as a dashed line. The inset shows the dependence of the peak currents on lactose concentration. Average normalized peak currents and SE are given. An apparent KD of 17 ( 3 mM is obtained from the hyperbolic fit (red trace). (B) Rate constants determined from the transient currents at different lactose concentrations at pH 5.2. From the transient currents, the rate constants were calculated with an iterative least-squares deconvolu- tion algorithm (17, 22). Average rate constants and SE from three or four data sets at a given lactose concentration are given. A hyperbolic fit to the data yields values for the forward (k2þ = 181 ( 14 s-1) and reverse (k2- = 33 ( 5 s-1) rate constants, as well as for the apparent KD (23 ( 6 mM) (see eq 2). constant currents indicating that the pH value in the proteolipo- somes had indeed adjusted to the external pH. The current traces fall into three different classes (Figure 1A). (1) At alkaline pH (7.6 and 8.5 for lactose), the magnitudes of the signals decrease with a decay time from peak to half-maximal current (τ1/2) of more than 20 ms. These transient currents are dominated by steady-state electrogenic downhill sugar/Hþ sym- port, and their decay is due to charging of the liposomal membrane by the transport activity of LacY (17). In this pH range, the peak currents recorded with lactose are larger than with melibiose, in agreement with lactose being the substrate transported with the highest turnover (17). (2) At intermediate pH values (7.0 and 6.3 for lactose), the transient currents are clearly biphasic. The improvement in time resolution allows resolution of a double peak (pH 7.0 for lactose). This double peak is even more pronounced with a 100 mM melibiose con- centration jump at pH 7.6. A double peak can only be explained