Exploring the P-glycoprotein binding cavity with polyoxyethylene alkyl ethers

Abstract

P-glycoprotein (ABCB1) moves allocrits from the cytosolic to the extracellular membrane leaflet, preventing their intrusion into the cytosol. It is generally accepted that allocrit binding from water to the cavity lined by the transmembrane domains occurs in two steps, a lipid-water partitioning step, and a cavity-binding step in the lipid membrane, whereby hydrogen-bond (i.e., weak electrostatic) interactions play a crucial role. The remaining key question was whether hydrophobic interactions also play a role for allocrit binding to the cavity. To answer this question, we chose polyoxyethylene alkyl ethers, C(m)EO(n), varying in the number of methylene and ethoxyl residues as model allocrits. Using isothermal titration calorimetry, we showed that the lipid-water partitioning step was purely hydrophobic, increasing linearly with the number of methylene, and decreasing with the number of ethoxyl residues, respectively. Using, in addition, ATPase activity measurements, we demonstrated that allocrit binding to the cavity required minimally two ethoxyl residues and increased linearly with the number of ethoxyl residues. The analysis provides the first direct evidence, to our knowledge, that allocrit binding to the cavity is purely electrostatic, apparently without any hydrophobic contribution. While the polar part of allocrits forms weak electrostatic interactions with the cavity, the hydrophobic part seems to remain associated with the lipid membrane. The interplay between the two types of interactions is most likely essential for allocrit flipping.

Figure 1

Free energies of binding of C₁₂EO_n (n = 3–10, 23) to membrane and transporter as a function of the number of ethoxyl groups (n) (T = 37°C). (A) Free energy of lipid-water partitioning, $\Delta G_{\text{lw}}^0$ (▴); free energy of transporter-water binding (first binding region) $\Delta G_{\text{tw}\left(1\right)}^0$ (□), free energy of transporter-water binding (second binding region) $\Delta G_{\text{tw}\left(\text{2}\right)}^0$ (○), both derived from ATPase activity measurements. (B) Free energy of transporter-lipid binding (first binding region) $\Delta G_{\text{tl}\left(1\right)}^0$ (■), (second binding region) $\Delta G_{\text{tw}\left(\text{2}\right)}^0$ (●). Linear fits were performed in the range of n = 4–10. Values for $\Delta G_{\text{tw}\left(1\right)}^0$ and $\Delta G_{\text{tw}\left(\text{2}\right)}^0$ are the averages of at least five measurements.

Figure 2

Free energies of binding of C_mEO₈ (m = 10, 12, 14) to the membrane and to the transporter as a function of the number of methylene groups (m). Free energy of lipid-water partitioning, $\Delta G_{\text{lw}}^0$ (▴) (measured at T = 25°C and calculated at 37°C), free energy of transporter-water binding (first binding region) $\Delta G_{\text{tw}\left(1\right)}^0$ (▪), and free energy of transporter-water binding (second binding region) $\Delta G_{\text{tw}\left(\text{2}\right)}^0$ (●) (T = 37°C).

Figure 3

P-gp ATPase activity measured as a function of concentration of C₁₂EOn (n = 3–10, 23) in plasma membrane vesicles of NIH-MDR1-G185 cells (T = 37°C and pH 7.0). C₁₂EO₃ (▪), C₁₂EO₄ (★), C₁₂EO₅ (), C₁₂EO₆ (▴), C₁₂EO₇ (), C₁₂EO₈ (●), C₁₂EO₉ (♦), C₁₂EO₁₀ (◂), and C₁₂EO₂₃ (○). Data are expressed as the average of two measurements. (Solid lines) Fits to Eq. 4.

Figure 4

P-gp ATPase activity measured as a function of concentration of C_mEO₈ (m = 10, 12, 14) in plasma membrane vesicles of NIH-MDR1-G185 cells (T = 37°C and pH 7.0). C₁₀EO₈ (▪), C₁₂EO₈ (●), and C₁₄EO₈ (♦). Data are expressed as the average of two measurements. (Solid lines) Fits to Eq. 4.

Figure 5

Detergent mole fraction X₍₁₎ (▪) and X₍₂₎ (○) at the concentration of half-maximum activation, K₍₁₎, and inhibition, K₍₂₎, as a function of the number of ethoxyl groups, n, at 37°C.

Figure 6

Difference in the free energy of binding from water to the activating and inhibitory binding region of P-gp, $\Delta G_{\text{tw}\left(\text{2}\right)}^0$ – $\Delta G_{\text{tw}\left(1\right)}^0$, as a function of the cross-sectional area, A_D, of allocrits, determined by surface activity measurements. Drugs measured previously (Fig. 6 in (16)) (○). E(n) corresponds to C₁₂EO_n (n = 3, 4, 5, 8, 10, 23) (■).