A multidrug ABC transporter with a taste for salt.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter

Arginine-482 is not essential for transport of antibiotics, primary bile acids and unconjugated sterols by the human breast cancer resistance protein (ABCG2)

The human BCRP (breast cancer resistance protein, also known as ABCG2) is an ABC (ATP-binding cassette) transporter that extrudes various anticancer drugs from cells, causing multidrug resistance. To study the molecular determinants of drug specificity of BCRP in more detail, we have expressed wild-type BCRP (BCRP-R) and the drug-selected cancer cell line-associated R482G (Arg(482)→Gly) mutant BCRP (BCRP-G) in Lactococcus lactis. Drug resistance and the rate of drug efflux in BCRP-expressing cells were proportional to the expression level of the protein and affected by the R482G mutation, pointing to a direct role of BCRP in drug transport in L. lactis. In agreement with observations in mammalian cells, the BCRP-R-mediated transport of the cationic substrates rhodamine 123 and tetramethylrosamine was significantly decreased compared with the activity of BCRP-G. In addition, BCRP-R showed an enhanced interaction with the anionic anticancer drug methotrexate when compared with BCRP-G, suggesting that structure/substrate specificity relationships in BCRP, as observed in eukaryotic expression systems, are maintained in prokaryotic L. lactis. Interestingly, BCRP-R exhibited a previously unestablished ability to transport antibiotics, unconjugated sterols and primary bile acids in L. lactis, for which the R482G mutation was not critical. Since Arg(482) is predicted to be present in the intracellular domain of BCRP, close to transmembrane segment 3, our results point to a role of this residue in electrostatic interactions with charged substrates including rhodamine 123 and methotrexate. Since unconjugated sterols are neutral molecules and bile acids and many antibiotics are engaged in protonation/deprotonation equilibria at physiological pH, our observations may point either to a lack of interaction between Arg(482) and neutral or neutralized moieties in these substrates during transport or to the interaction of these substrates with regions in BCRP not including Arg(482)

Drug-Lipid A Interactions on the Escherichia coli ABC Transporter MsbA

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K(i) values of 16 and 11 μM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K(i) of 57 μM, in a similar range as the K(i) for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005)

A multidrug ABC transporter with a taste for salt.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter

Ion-coupled transport in proteoliposomes.

<p>A, B, ³⁶Cl⁻ uptake (100 µM) by LmrA-MD (•), E314A LmrA-MD (⋄), EE LmrA-MD (▪) or empty liposomes (▵) in the presence of a Δψ (interior negative) of −120 mV (A) or -ZΔpH (interior alkaline) of −49 mV (B). In the duplicate experiment for LmrA-MD (□) in (B), the addition of uncoupler (valinomycin plus nigericin, 1 µM each) at the arrow resulted in efflux of accumulated ³⁶Cl⁻, indicating concentrative uptake of the ion. C, ΔpH (interior alkaline)-dependent ³⁶Cl⁻ uptake by LmrA (•), EE LmrA (▪) or empty liposomes (▵). D, ΔpH (interior alkaline)-dependent uptake of non-radioactive Cl⁻ (1 mM) by LmrA is observed as a quench in the fluorescence of the SPQ fluorophore trapped in the lumen of the proteoliposomes. Quenching was also observed in empty liposomes (control) in the presence of the Cl⁻/OH⁻ antiporter TBT-Cl (1 µM). E, Kinetic analysis of ΔpH (interior alkaline)-dependent ³⁶Cl⁻ uptake by LmrA. F, ΔpH (interior alkaline)-dependent uptake of ²²Na (25 µM) by LmrA-MD. G, Uptake of unlabelled Na⁺ (10 mM) by LmrA was detected as an increase in the fluorescence of the membrane-impermeable sodium green probe trapped in the lumen. H, Na⁺ (100 µM) stimulates the ΔpH-dependent uptake of ³⁶Cl⁻ (100 µM) by LmrA compared to control containing 99 µM NMG⁺ plus 1 µM Na⁺. I, H⁺ efflux in proteoliposomes loaded with pH probe BCECF in the presence of an outwardly directed NaCl gradient. Control, empty liposomes. (<i>n</i> = 5)</p