Interaction of LDS-751 and rhodamine 123 with P-glycoprotein: evidence for simultaneous binding of both drugs.
The P-glycoprotein efflux pump, an ABC superfamily member, can export a wide variety of hydrophobic drugs, natural products, and peptides from cells, powered by the energy of ATP hydrolysis. Transport substrates appear to first partition into the membrane and then interact with the protein within the cytoplasmic leaflet. Two drug binding sites within P-glycoprotein have been described which interact allosterically, the H-site (binds Hoechst 33342) and the R-site (binds rhodamine 123); however, the structural and functional relationship between the various binding sites appears complex. In this work, we have used fluorescence spectroscopic approaches to characterize the interaction of the transporter with LDS-751 and rhodamine 123, both of which are believed to bind to the putative R-site based on functional transport studies. By carrying out single and sequential dual fluorescence titrations of purified P-glycoprotein with the two substrates, we observed that bound LDS-751 interacted with bound rhodamine 123. Rhodamine 123 and LDS-751 showed a reciprocal negative interaction, each reducing the binding affinity of the other by 5-fold, indicating that the two compounds were simultaneously bound to the protein to form a ternary complex. Fitting of the dependence of the apparent Kd for LDS-751 binding on rhodamine 123 concentration suggested that the two compounds interacted noncompetitively. We conclude that the two-site drug binding model for P-glycoprotein requires modification. The putative R-site appears large enough to accommodate two compounds simultaneously. The locations where LDS-751 and rhodamine 123 bind are likely adjacent to each other, possibly overlapping, and may be within a hydrophobic pocket.
Lugo
Miguel R
MR
Instituto de Biología Experimental, Facultad de Ciencias, Universidad Central de Venezuela, Caracas, Venezuela.
Sharom
Frances J
FJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000378:metabolism*; D009930:Organic Chemicals / Q000378:metabolism; D011485:Protein Binding; D020112:Rhodamine 123 / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C057718:6-(Dimethylamino)-2-((1E,3E)-4-(4-(dimethylamino)phenyl)-1,3-butadien-1-yl)-1-ethylquinolinium perchlorate; D020112:Rhodamine 123