Therapeutics , Targets , and Chemical Biology MDR 1 Synonymous Polymorphisms Alter Transporter Speci fi city and Protein Stability in a Stable Epithelial Monolayer

The drug efflux function of P-glycoprotein (P-gp) encoded byMDR1 can be influenced by genetic polymorphisms, including two synonymous changes in the coding region ofMDR1. 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 similarMDR1 DNA copy number were developed and termed LLC-MDR1WT (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. Cancer Res; 74(2); 598–608. 2013 AACR.