title: MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer.
abstract: The drug efflux function of P-glycoprotein (P-gp) encoded by MDR1 can be influenced by genetic polymorphisms, including two synonymous changes in the coding region of MDR1. Here we report that the conformation of P-gp and its drug efflux activity can be altered by synonymous polymorphisms in stable epithelial monolayers expressing P-gp. Several cell lines with similar MDR1 DNA copy number were developed and termed LLC-MDR1-WT (expresses wild-type P-gp), LLC-MDR1-3H (expresses common haplotype P-gp), and LLC-MDR1-3HA (a mutant that carries a different valine codon in position 3435). These cell lines express similar levels of recombinant mRNA and protein. P-gp in each case is localized on the apical surface of polarized cells. However, the haplotype and its mutant P-gps fold differently from the wild-type, as determined by UIC2 antibody shift assays and limited proteolysis assays. Surface biotinylation experiments suggest that the non-wild-type P-gps have longer recycling times. Drug transport assays show that wild-type and haplotype P-gp respond differently to P-gp inhibitors that block efflux of rhodamine 123 or mitoxantrone. In addition, cytotoxicity assays show that the LLC-MDR1-3H cells are more resistant to mitoxantrone than the LLC-MDR1-WT cells after being treated with a P-gp inhibitor. Expression of polymorphic P-gp, however, does not affect the host cell's morphology, growth rate, or monolayer formation. Also, ATPase activity assays indicate that neither basal nor drug-stimulated ATPase activities are affected in the variant P-gps. Taken together, our findings indicate that "silent" polymorphisms significantly change P-gp function, which would be expected to affect interindividual drug disposition and response.
authors.affiliation: Authors' Affiliations: Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH; and Center for Biologics Evaluation and Research, Division of Hematology, Food and Drug Administration, Bethesda, Maryland.
authors.forename: King Leung
authors.initials: KL
authors.lastname: Fung
authors.forename: James
authors.initials: J
authors.lastname: Pan
authors.forename: Shinobu
authors.initials: S
authors.lastname: Ohnuma
authors.forename: Paul E
authors.initials: PE
authors.lastname: Lund
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Pixley
authors.forename: Chava
authors.initials: C
authors.lastname: Kimchi-Sarfaty
authors.forename: Suresh V
authors.initials: SV
authors.lastname: Ambudkar
authors.forename: Michael M
authors.initials: MM
authors.lastname: Gottesman
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D019921:Biotinylation; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019008:Drug Resistance, Neoplasm; D015972:Gene Expression Regulation, Neoplastic; D006239:Haplotypes; D006801:Humans; D008942:Mitoxantrone / Q000737:chemistry; D009154:Mutation; D011110:Polymorphism, Genetic; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D020112:Rhodamine 123 / Q000737:chemistry; D013552:Swine
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D011994:Recombinant Proteins; D020112:Rhodamine 123; D008942:Mitoxantrone; D000251:Adenosine Triphosphatases