MDR1 synonymous polymorphisms alter transporter specificity and protein stability in a stable epithelial monolayer

Area of Science:

• Pharmacogenomics
• Molecular Biology
• Cell Biology

Background:

• The drug efflux pump P-glycoprotein (P-gp), encoded by the MDR1 gene, plays a crucial role in drug disposition.
• Genetic polymorphisms in MDR1 can influence P-gp function, but the impact of synonymous variations remains less understood.

Purpose of the Study:

• To investigate how synonymous polymorphisms in the MDR1 gene affect P-gp conformation, drug efflux activity, and cellular response to drugs.
• To characterize the functional consequences of specific MDR1 synonymous variants in a cellular model.

Main Methods:

• Development of stable epithelial cell lines expressing wild-type, haplotype, and mutant P-gp with similar expression levels.
• Utilized UIC2 antibody shift assays, limited proteolysis, and surface biotinylation to assess P-gp conformation and trafficking.
• Performed drug transport assays with rhodamine 123 and mitoxantrone, alongside cytotoxicity assays and ATPase activity measurements.

Main Results:

• Synonymous polymorphisms alter P-gp conformation and increase its recycling time, leading to differential drug efflux.
• Cell lines expressing variant P-gp showed altered responses to P-gp inhibitors and increased resistance to mitoxantrone.
• Basal and drug-stimulated ATPase activities, cell morphology, growth rate, and monolayer formation were not affected by P-gp variants.

Conclusions:

• Synonymous MDR1 polymorphisms significantly impact P-gp function and drug transport, despite being traditionally considered
• silent.
• These findings suggest that synonymous variants can influence interindividual drug disposition and therapeutic outcomes.
• The study underscores the importance of considering synonymous polymorphisms in pharmacogenetic studies and drug development.