P-glycoprotein modulates the fluidity gradient of the plasma membrane of multidrug resistant CHO cells

Abstract

Cryo-electron microscopy has yielded high-resolution structural data of the multidrug efflux transporter P-glycoprotein (ABCB1), but its direct and indirect interactions within the native membrane environment have remained largely unexplored. Here, we compared the fluidity gradients of plasma membranes of the drug-sensitive CHO cell line AuxB1 and its P-glycoprotein overexpressing derivative B30 by fluorescence anisotropy of embedded n-(9-anthroyloxy) fatty acid probes (n = 2, 7, 9, 12, 16) in the temperature range of 10-50 °C. The shape of the temperature profiles of probe mobility was comparable in AuxB1 and B30 membranes, but did not match. Overexpression of P-glycoprotein smoothened the transversal gradient of the out-of-plane mode of rotation of the probes, which may facilitate the partitioning of hydrophobic drugs into the membrane and thereby increase the speed of P-glycoprotein to pump the drug out of the cell.

Keywords: ABC transporter; ABCB1; P‐glycoprotein; membrane fluidity; multidrug resistance; n‐(9‐anthroyloxy) fatty acid.

Fig. 1

Plot of Y = f(X) for 2‐AS in DPPC vesicles at 37 °C deduced from three independent vesicle preparations. Y and X parameters are described in Material and methods in the section ‘Data analysis’ [Eqns (1) and (2)].

Fig. 2

Plot of Y = f(X) for 2‐AS in AuxB1 (solid circles) and CH^RB30 (solid squares) plasma membranes at 20 °C (n = 3 technical replicates). Y and X parameters are described in Material and methods in the section ‘Data analysis’ [Eqns (1) and (2)].

Fig. 3

Transversal gradient of the rotation correlation coefficient R _op, the out‐of‐plane rotation around the ester bond at C9 of the anthracene ring of n‐(9‐anthroyloxy) fatty acid probes (n = 2, 7, 9, 12, 16) embedded into plasma membranes of the drug‐sensitive CHO cell line AuxB1 (solid line) and of its highly multidrug‐resistant derivative CH^RB30 (dashed line) within the range of 10–50 °C. Heating curves were recorded for each probe and each cell line in triplicate.

Fig. 4

Transversal gradient of the rotation correlation coefficient R _ip, the in‐plane acyl chain segmental reorientation motion of n‐(9‐anthroyloxy) fatty acid probes (n = 2, 7, 9, 12, 16) embedded into plasma membranes of the drug‐sensitive CHO cell line AuxB1 (solid line) and of its highly multidrug‐resistant derivative CHRB30 (dashed line) within the range of 10–50 °C. Heating curves were recorded for each probe and each cell line in triplicate.

Fig. 5

Single recordings of the fluorescence anisotropy of 2‐AS at 381 nm embedded in AuxB1 (gray dots, yellow dots) and CH^RB30 (orange dots, blue dots) plasma membranes in the absence (gray dots, orange dots) and presence (yellow dots, blue dots) of 10 μm verapamil within the range of 10–50 °C. The dots represent the individual measurements at 381 nm during the recording of the heating curves.

Fig. 6

Rotation correlation coefficients R _op (gray and yellow lines) and R _ip (blue and orange lines) of the probe 2‐AS embedded in CH^RB30 plasma membranes in the absence (gray and blue lines) and presence (yellow and orange lines) of 10 μm verapamil within the range of 10–50 °C (n = 2 technical replicates).