Research ArticleBIOCHEMISTRY

Powering the ABC multidrug exporter LmrA: How nucleotides embrace the ion-motive force

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Science Advances  19 Sep 2018:
Vol. 4, no. 9, eaas9365
DOI: 10.1126/sciadv.aas9365
  • Fig. 1 Current-voltage responses for different buffer compositions.

    LmrA-WT–containing phospholipid bilayers were subjected to a voltage step protocol with 10-mV steps for 1 s (ranging from −100 to +100 mV) from a holding potential of 0 mV. Macroscopic currents were recorded in symmetric buffer solutions containing 10 mM HEPES, 20 mM EGTA, and 10 mM NaCl (pH 7.2, NaOH) (blue triangle) or solutions containing asymmetric salt concentrations of 10 mMin/100 mMout NaCl (red circle), LiCl (purple square), KCl (black triangle), or equimolar Na2SO4 (green diamond). Data points (mean ± SEM) are based on at least three independent measurements.

  • Fig. 2 Ion conductance in response to nucleotide binding.

    (A to F) Macroscopic current responses to the imposition of the same voltage protocol as in Fig. 1, in symmetric 10 mM HEPES, 20 mM EGTA, and 10 mM NaCl solutions (pH 7.2, NaOH) in the absence or presence of 5 mM Mg-nucleotide in the external buffer. Scale bars in (A) are applicable to all current responses. (G) Determination of ion stoichiometry from Erev measurements. Erev was measured in the presence of 5 mM Mg-ATP as a function of the log10 of the external concentration of single buffer components: Na+ (black circle), Cl (blue square), or monovalent cationic HEPES+ (red diamond). Data points (±SEM) show the mean Erev for at least three independent measurements. The internal concentration of Na+ and Cl was maintained at 60 mM, and the external concentration was raised from 60 to 100, 150, or 200 mM, with HEPES maintained at 10 mM in internal and external solutions. To measure the effect of HEPES (pH 6.5), the internal solution contained 60 mM NaCl, 20 mM EGTA, and 10 mM HEPES. The external HEPES concentration was raised from 10 to 25, 100, or 125 mM, yielding HEPES+ concentrations of 0.29, 0.73, 2.9, and 3.6 μM. The ion stoichiometry was determined from the slope of the linear fit using the Nernst equilibrium equation (data analysis S1). The slope values for Na+, Cl, and HEPES+ were 104.6 ± 5.3 mV, −59.1 ± 7.8 mV, and −51.1 ± 4.9 mV, respectively (n = 3). (H) Current-voltage relation (I-V) response graph for LmrA-WT using symmetric solutions as listed under (A) with 5 mM Mg-ATP in the external buffer (blue trace), or asymmetric solutions containing [Na+]in/[Na+]out = 50 mM/100 mM and [Cl]in/[Cl]out = 50 mM/50 mM in the presence of the ATP (black trace), or containing [NaCl]in/[NaCl]out = 10 mM/150 mM without nucleotide (red trace). The Erev for the blue, black, and red traces was −2.0 ± 1.2 mV, 37.6 ± 1.5 mV, and 66.7 ± 6.1 mV (n = 3), respectively.

  • Fig. 3 Ion conductance is initiated by Mg-ATP binding and terminated by ATP hydrolysis.

    (A to D) Single-transporter current responses to the imposition of a membrane potential of −50 mV with symmetric solutions containing 10 mM NaCl, 50 mM KCl, 10 mM HEPES, and 20 mM EGTA (pH 7.2, NaOH) in the presence of Mg-ATP in the external buffer. Red dashed lines refer to nonactive and active states (usually referred to as closed and open states in channel studies). All-point amplitude histograms presented on the right of every recording were obtained using an equivalent current range for all cases (bin count = 400). Mg-ATP fails to induce single-transporter conductance for transport-inactive mutants LmrA-E314E and LmrA-EE, which were inserted in the bilayer at a similar protein concentration as LmrA-WT. (E to H) Analyses of nonactive and active time distributions from which relevant parameters are derived [*P < 0.05, ***P < 0.001, one-way analysis of variance (ANOVA)]. Inhibition of ATP hydrolysis prolongs the time period during which ion exchange is observed.

  • Fig. 4 Ion exchange is linked to drug transport.

    (A and B) Transport of ethidium (2 μM) in DNA-loaded proteoliposomes containing LmrA-WT (A) or ATPase-deficient LmrA-ΔK388 or proton transport–deficient LmrA-E314A (B) in an inside-out fashion, in the absence or presence of nucleotide and/or the stepwise imposition of a sodium-motive force ΔpNa (interior positive and [Na+]in/([Na+]out = 100 mM/1 mM), chemical proton gradient ΔpH (pHin 8.0/pHout 6.8), and chemical chloride gradient ΔpCl ([Cl]in/([Cl]out = 50 mM/150 mM). Line colors are the same as bar colors and refer to the same experimental conditions. (C to E) Effect of salts on the ethidium accumulation rate in cells with (black circles) and without LmrA (white circles) as a percentage of the rate in the absence of salts. (F) Effect of 100 mM NaCl in the external buffer on ethidium efflux from cells preloaded with 2 μM ethidium. Efflux was initiated by the addition of 25 mM glucose (+Glc) as a source of metabolic energy. (G and H) Growth of L. lactis expressing LmrA-WT (blue trace) or LmrA-ΔK388 (red trace) in the presence of 4 μM ethidium in standard M17 medium (pH 7.0) (G) or M17 medium containing 16.7 mM NaOH (pH 7.5) plus 25 mM NaCl (H). The error bars for some of the data points are too small to be displayed and are hidden behind the data point symbols. Data represent observations in three or more independent experiments with independently prepared batches of proteoliposomes and cells. Values in histograms show significance of fluorescence levels at steady state and are expressed as means ± SEM (***P < 0.001, ****P < 0.0001, one-way ANOVA).

  • Fig. 5 N137A mutation impairs drug and ion transport by LmrA.

    (A) Ethidium efflux from preloaded cells containing LmrA-N137A was performed as described in Fig. 4F and was initiated by the addition of 25 mM glucose (+Glc) as a source of metabolic energy. Transport in the absence or presence of 50 mM Na2SO4 is compared with the Na+-stimulated activity for LmrA-WT. Line colors are the same as bar colors and refer to the same experimental conditions. (B) Ethidium transport in proteoliposomes was measured with artificial imposition of the same ion gradients as described in Fig. 4 (A and B). Where indicated, LmrA-WT activity was inhibited by inclusion of 1 mM Na-vanadate in the assay buffer. (C) H+ efflux was measured in cells loaded with pH probe 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFDASE) to monitor the intracellular pH in the absence or presence of 125 mM Na2SO4 in the external buffer (based on up to nine independent measurements). Metabolic energy was generated in the cells by the addition of glucose at t = 0 min. (D) Erev was measured as described in Fig. 2G as a function of the log10 of the external Na+ concentration. The slope value was 58.8 ± 1.6 mV. Values in histograms show significance of fluorescence end levels and are expressed as means ± SEM (**P < 0.01, ****P < 0.0001, one-way ANOVA).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/9/eaas9365/DC1

    Fig. S1. Expression and purification of LmrA proteins.

    Fig. S2. Peptide coverage maps from the Mascot LC-MS/MS database search results.

    Fig. S3. Ion binding sites in LmrA.

    Fig. S4. Binding sites for Na+ and Cl in example proteins.

    Fig. S5. Expression and purification of LmrA-N137A mutant protein.

    Fig. S6. Conservation of residue N137 in ABC multidrug transporters.

    Table S1. Mascot search results for mass spectrometry data for purified LmrA-WT.

    Table S2. Mascot search results for mass spectrometry data for purified LmrA-ΔK388.

    Table S3. Speciation of HEPES at pH 6.5 as a function of the HEPES concentration.

    Data analysis S1. Determination of Erev values and ion stoichiometry.

    Data analysis S2. Comparisons of ion transport models.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Expression and purification of LmrA proteins.
    • Fig. S2. Peptide coverage maps from the Mascot LC-MS/MS database search results.
    • Fig. S3. Ion binding sites in LmrA.
    • Fig. S4. Binding sites for Na+ and Cl in example proteins.
    • Fig. S5. Expression and purification of LmrA-N137A mutant protein.
    • Fig. S6. Conservation of residue N137 in ABC multidrug transporters.
    • Table S1. Mascot search results for mass spectrometry data for purified LmrA-WT.
    • Table S2. Mascot search results for mass spectrometry data for purified LmrA-ΔK388.
    • Table S3. Speciation of HEPES at pH 6.5 as a function of the HEPES concentration.
    • Data analysis S1. Determination of Erev values and ion stoichiometry.
    • Data analysis S2. Comparisons of ion transport models.

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