Kinetic Effects of Inside‐Positive Membrane Potential on the Lactose Efflux of LacY

Abstract

LacY, the lactose permease of E. coli., utilizes the free energy released from the downhill movement of H+ in response of an electrochemical H+ gradient (Δμ̃H+, interior negative and/or alkaline) to catalyze the accumulation of lactose against a concentration gradient. In the absence of Δμ̃H+, the downhill transport of lactose in response of a concentration gradient drives uphill translocation of H+ to generate a H+ gradient, and the polarity of the H+ gradient depends on the direction of lactose gradient. In other words, LacY is a lactose/H+ symporter, in which the lactose transport and H+ translocation are obligatorily coupled. LacY functions as a monomer with a total of 12 transmembrane helices arranged into two 6‐helix bundles tethered by a long loop between helix VI and VII. The sugar‐ and H+‐binding sites are located at approximate the center of the permease and can be accessed from either side of the membrane via an alternating access conformational change driven by the binding and dissociation of sugar. In LacY, Glu325 plays an essential role in H+ translocation since replacing Glu325 with neutral amino residues abolishes transport activities involving H+ translocation, but the mutants still bind sugar with normal affinity and catalyze equilibrium exchange. Previous studies have shown that the presence of Δμ̃H+ (interior negative) significantly accelerates lactose influx by decreasing the Km 50‐100‐fold with no change in Kd. The goal of this study is to investigate kinetic effects of Δμ̃H+ on lactose efflux in right‐side‐out membrane vesicles. We measured the initial rate of efflux at different pHs in the absence or presence of an imposed membrane potential (ΔΨ, generated by K+ influx in response of a concentration gradient in the presence of valinomycin, interior positive). The results revealed that the lactose efflux of WT LacY exhibits an apparent pK value near 7.2, which is not affected by the imposition of ΔΨ (interior positive). The initial rate of lactose efflux increases in the presence of imposed ΔΨ (interior positive) at all tested pHs, although enhancement is about two orders of magnitude less than that observed in lactose influx. Moreover, the effect of ΔΨ (interior positive) on the Km of lactose efflux is almost negligible compared to the dramatic change of the Km of lactose influx caused by ΔΨ (interior negative). In contrast to WT LacY, the E325A mutant does not carry out lactose efflux in the absence or presence of imposed ΔΨ (interior positive) at all tested pHs.Support or Funding InformationNSF Eager Grant MCB‐1547801 to H.R.K., NIH Grant 1R01 GM120043 to H.R.K., and a grant from Ruth and Bucky Stein to H.R.K..