A multidrug ABC transporter with a taste for salt

Abstract

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.

Figure 1. Mass spectra of purified LmrA and LmrA-MD showing predominant homodimer formation for both proteins.

Peaks assigned to binding of one cardiolipin molecule are labelled (blue stars), and measured molecular masses are shown.

Figure 2. Electrophysiological analyses of ion transport in proteoliposomes by ‘dip-tip’ method.

A, Voltage step protocol from a holding potential of 0 mV to various voltages (ranging from −105 mV to +115 mV), back to 0 mV. B, C, D, Current traces for LmrA-MD (B) or empty liposomes without protein (C) or EE LmrA-MD (D) in the presence of 10 mM NaCl. E, F, Current traces for LmrA-MD in the presence of SO₄ ²⁻ instead of Cl⁻ (E), or NMG⁺ instead of Na⁺ and K⁺ (F). G, I–V curves from the traces in (B–F) (○, LmrA-MD; •, replacement of Cl⁻ by SO₄ ²⁻; □, replacement of K⁺/Na⁺ by NMG⁺; ▿, EE; ▵, empty liposomes; the latter two traces are hidden behind •). (n = 15)

Figure 3. Ion-coupled transport in proteoliposomes.

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. (n = 5)

Figure 4. Ion transport in intact cells.

A, Cl⁻ efflux in cells preloaded with Na³⁶Cl (100 µM) upon the addition of glucose (▪, LmrA; □, non-expressing control; ⧫, EE LmrA; ○, ΔK388 LmrA). B, Effect of the concentration of Na⁺, NMG⁺ or Cl⁻ on the ATPase activity of purified LmrA (open symbols) or LmrCD (•) measured at 2 mM Mg-ATP. C,D,E, H⁺ efflux in energized cells, loaded with pH probe CFDASE to monitor the intracellular pH (pH_in) in the absence (C) or presence of (D) 0.25 M NaCl or (E) 0.672 M sucrose in the external buffer (each equivalent to 521 mOsm) (▪, LmrA; □, non-expressing control; ⧫, EE LmrA; •, E314A LmrA; ▴, ΔK388 LmrA). Metabolic energy was generated in the cells by the addition of 20 mM glucose (at t = 0 min in the figures), 15 min after the addition of the NaCl or sucrose or solvent control. (n = 8)

Figure 5. LmrA activity enhances cell survival.

A, Viability of energized cells (solid bar, LmrA; grey bar, non-expressing control) adapted for 30 min in buffer containing 0.5 M sucrose without or with 100 mM NaCl, 100 mM KCl or 50 mM Na₂SO₄, followed by 100-fold dilution into ultrapure water, or in buffer containing 0.125 M sucrose and 25 mM NaCl (control referred to as 4-fold dilution). B, Effect of mutations in LmrA on the viability after dilution of cells pre-exposed to 100 mM NaCl plus 0.5 M sucrose under conditions as in (A). (n = 10)