title: Localization and substrate selectivity of sea urchin multidrug (MDR) efflux transporters.
abstract: In this study, we cloned, expressed and functionally characterized Stronglycentrotus purpuratus (Sp) ATP-binding cassette (ABC) transporters. This screen identified three multidrug resistance (MDR) transporters with functional homology to the major types of MDR transporters found in humans. When overexpressed in embryos, the apical transporters Sp-ABCB1a, ABCB4a, and ABCG2a can account for as much as 87% of the observed efflux activity, providing a robust assay for their substrate selectivity. Using this assay, we found that sea urchin MDR transporters export canonical MDR susbtrates such as calcein-AM, bodipy-verapamil, bodipy-vinblastine, and mitoxantrone. In addition, we characterized the impact of nonconservative substitutions in the primary sequences of drug binding domains of sea urchin versus murine ABCB1 by mutation of Sp-ABCB1a and treatment of embryos with stereoisomeric cyclic peptide inhibitors (QZ59 compounds). The results indicated that two substitutions in transmembrane helix 6 reverse stereoselectivity of Sp-ABCB1a for QZ59 enantiomers compared with mouse ABCB1a. This suggests that subtle changes in the primary sequence of transporter drug binding domains could fine-tune substrate specificity through evolution.
authors.affiliation: Marine Biology Research Division, Scripps Institution of Oceanography, University of California San Diego, La Jolla, California 92093, USA.
authors.forename: Tufan
authors.initials: T
authors.lastname: Gökirmak
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Campanale
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Shipp
authors.forename: Gary W
authors.initials: GW
authors.lastname: Moy
authors.forename: Houchao
authors.initials: H
authors.lastname: Tao
authors.forename: Amro
authors.initials: A
authors.lastname: Hamdoun
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010456:Peptides, Cyclic / Q000494:pharmacology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D047270:Strongylocentrotus purpuratus / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D026901:Membrane Transport Proteins; D010456:Peptides, Cyclic; C539646:cyclic-tris-(R)-valineselenazole