The reconstituted P-glycoprotein multidrug transporter is a flippase for glucosylceramide and other simple glycosphingolipids

Abstract

The Pgp (P-glycoprotein) multidrug transporter, which is linked to multidrug resistance in human cancers, functions as an efflux pump for non-polar drugs, powered by the hydrolysis of ATP at its nucleotide binding domains. The drug binding sites of Pgp appear to be located within the cytoplasmic leaflet of the membrane bilayer, suggesting that Pgp may function as a 'flippase' for hydrophobic compounds. Pgp has been shown to translocate fluorescent phospholipids, and it has been suggested that it may also interact with GlcCer (glucosylceramide). Here we use a dithionite fluorescence quenching technique to show that reconstituted Pgp can flip several NBD (nitrobenzo-2-oxa-1,3-diazole)-labelled simple glycosphingolipids, including NBD-GlcCer, from one leaflet of the bilayer to the other in an ATP-dependent, vanadate-sensitive fashion. The rate of NBD-GlcCer flipping was similar to that observed for NBD-labelled PC (phosphatidylcholine). NBD-GlcCer flipping was inhibited in a concentration-dependent, saturable fashion by various Pgp substrates and modulators, and inhibition correlated well with the Kd for binding to the protein. The addition of a second sugar to the headgroup of the glycolipid to form NBD-lactosylceramide drastically reduced the rate of flipping compared with NBD-PC, probably because of the increased size and polarity contributed by the additional sugar residue. We conclude that Pgp functions as a broad-specificity outwardly-directed flippase for simple glycosphingolipids and membrane phospholipids.

Figure 1. Structures of some of the NBD-labelled GSL used in this study

NBD–C₆-GlcCer, NBD–C₆-GalCer, NBD–C₆-LacCer and NBD–C₁₂-Cer are shown. NBD–C₁₂-LacCer has the NBD moiety attached via a 12-carbon chain, rather than the 6-carbon chain shown for NBD–C₆-LacCer.

Figure 2. Translocation of NBD-labelled lipids by Pgp in reconstituted proteoliposomes

(A) Proteoliposomes of egg PC containing Pgp and 0.3% (w/w) NBD–PC (16:0, 6:0) were incubated at 37 °C with 1 mM ATP and the regenerating system for various times. After addition of 0.2 mM vanadate, fluorescence emission (λ_ex=468 nm, λ_em=540 nm) was monitored at 22 °C until a stable baseline was achieved. After 3 min, 2 mM dithionite was added (indicated by an interruption in the trace), and after a stable baseline was again reached, 1% (w/v) Triton X-100 was added. Fluorescence traces were obtained at time zero with or without ATP (±ATP, 0 min) and 20 min after adding ATP (+ATP, 20 min). Traces were normalized to the fluorescence intensity recorded just prior to dithionite addition, which was taken as 100%. The vertical arrows represent the total fluorescence of NBD–PC in both the inner and outer leaflets (F_i+o), and the fluorescence of NBD–PC in the inner leaflet in the absence and presence of ATP (F_i). (B) Time course for the translocation of NBD–PC (16:0, 6:0) in proteoliposomes of egg PC containing reconstituted Pgp. Proteoliposomes were incubated for 0–90 min either with (●) or without (○) 1 mM ATP and the regenerating system, or with 1 mM ATP for the first 45 min, after which more ATP was added (2 mM final concentration) and the sample was incubated for a further 45 min (■). Data points represent the means for duplicate determinations; error bars represent the range (where not visible, they fall within the symbols). (C) Proteoliposomes containing Pgp and 0.3% (w/w) NBD–C₆-GlcCer were treated as in (A). (D) Translocation of NBD–PC (16:0, 6:0) inserted asymmetrically into the outer leaflet of egg PC proteoliposomes containing Pgp, with (●) or without (○) 1 mM ATP and the regenerating system.

Figure 3. Time course for the translocation of three NBD-labelled GSL and NBD–SM in proteoliposomes of egg PC containing reconstituted Pgp

Proteoliposomes were incubated either with (●) or without (○) 1 mM ATP and the regenerating system. The transbilayer distribution of the NBD–lipid was measured using dithionite quenching: (A) NBD–C₆-GlcCer, (B) NBD–C₆-GalCer, (C) NBD–C₆-LacCer and (D) NBD–C₆-SM. Data points represent the means±range for duplicate determinations; where not visible, the error bars fall within the symbols.

Figure 4. Characterization of Pgp-mediated translocation of NBD–C₆-GlcCer

(A) Dependence of translocation on the concentration of NBD–C₆-GlcCer in the bilayer. Reconstituted proteoliposomes of egg PC containing 0.1–1.0% (w/w) NBD–C₆-GlcCer were incubated for 20 min at 37 °C, and the extent of translocation was determined. Data were normalized to the extent of NBD–C₆-GlcCer translocated with 0.3% (w/w) NBD–lipid, which was taken as 100%, and are represented as the means±range for duplicate determinations. (B) Nucleotide dependence of NBD–C₆-GlcCer translocation. Egg PC proteoliposomes were incubated in the presence of 0, 1 or 2 mM ATP and the regenerating system, in the presence of 0.1 or 0.2 mM vanadate (Vi), 1 mM ADP or p[NH]ppA (AMP-PNP), and the transbilayer distribution of NBD–C₆-GlcCer was determined. The data were normalized to the value with 1 mM ATP, which was taken as 100%. Data points represent means±range for duplicate determinations. (C) ATP dependence of NBD–C₆-GlcCer translocation. Egg PC proteoliposomes were incubated at 37 °C for 20 min in the presence of 0–10 mM ATP and the regenerating system. The transbilayer distribution of NBD–C₆-GlcCer was determined, and the data were normalized to the value with 1 mM ATP, which was taken as 100%. Data points represent means±range for duplicate determinations.

Figure 5. Inhibition of Pgp-mediated NBD–C₆-GlcCer translocation by vanadate

Egg PC proteoliposomes were incubated in the presence of 1 mM ATP and the regenerating system plus 0–500 μM vanadate (●), or in the absence of ATP and the presence of 500 μM vanadate (○), and the transbilayer distribution of NBD–C₆-GlcCer was determined. Data were normalized to the value in the presence of 1 mM ATP but in the absence of vanadate, which was taken as 100%. Data points represent means±range for duplicate determinations; where not visible, error bars fall within the symbols.

Figure 6. Inhibition of ATP-dependent NBD–C₆-GlcCer translocation by various Pgp transport substrates and modulators

The net translocation of NBD–C₆-GlcCer was assessed in the presence of various drugs, relative to a control without drug (taken as 100%), and a control with drug but without ATP (taken as 0%). Proteoliposomes containing Pgp were treated with (A) leupeptin, (B) PSC-833, (C) colchicine or (D) verapamil, and translocation of NBD–C₆-GlcCer was assessed after 20 min at 37 °C. Data points represent means±range for duplicate determinations; where not visible, error bars fall within the symbols.

Figure 7. Quantification of inhibition of NBD–C₆-GlcCer translocation by cyclosporin A

(A) Net translocation over a 20 min time period was assessed using proteoliposomes containing Pgp in the presence of cyclosporin A, as in Figure 6. (B) Data from (A) displayed with a logarithmic scale for the independent variable. (C) Median effect plot of log(f_a/f_u) against log[cyclosporin A] (see the Experimental section for theory). D_m is determined from the intercept on the x axis, where log(f_a/f_u)=0, as indicated on the plot. Data points represent means±range for duplicate determinations; where not visible, error bars fall within the symbols.

Figure 8. Correlation between inhibition of NBD–C₆-GlcCer flippase activity and affinity of binding to Pgp for several transport substrates and modulators

Agents used were as follows: 1, PSC-833; 2, cyclosporin A; 3, verapamil; 4, leupeptin; 5, colchicine. The D_m values for the compounds are plotted on a log–log scale against their measured K_d values for binding to purified Pgp labelled with MIANS [2-(4-maleimidoanilino)-naphthalene-6-sulphonic acid].

Figure 9. Dithionite quenching of NBD–C₁₂-Cer in proteoliposomes

The bilayer distribution of egg PC proteoliposomes containing Pgp and 0.3% (w/w) NBD–C₁₂-Cer was determined at 22 °C using dithionite quenching as described in the legend to Figure 2. A stable plateau due to the fluorescence of inner-leaflet NBD–C₁₂-Cer could not be established. The bilayer distribution was also determined for egg PC liposomes lacking reconstituted Pgp, but including 0.15% (w/w) NBD–PC (16:0, 6:0) and 0.15% (w/w) NBD–C₁₂-Cer. Traces were normalized to the fluorescence intensity recorded just prior to the addition of dithionite. The vertical arrows represent the total fluorescence due to NBD-labelled lipids in both the inner and outer leaflets (F_i+o) and the fluorescence due to NBD–lipids in the inner leaflet (F_i).