MDR1 haplotypes significantly minimize intracellular uptake and transcellular P‐gp substrate transport in recombinant LLC‐PK1 cells

To date, research on the effect of single nucleotide polymorphisms (SNPs) on P‐glycoprotein (P‐gp) expression and functionality has rendered inconsistent results. This study systematically evaluates the impact of MDR1 haplotypes (1236/2677, 1236/3435, 2677/3435, 1236/2677/3435) on P‐gp functionality compared to individual SNPs (1236, 2677, and 3435) in validated stable recombinant epithelial cells. Recombinant LLC‐PK1 cells expressing MDR1wt or its variants were developed and validated for this purpose. Intracellular accumulation and time‐dependant efflux of a P‐gp substrate, Rhodamine 123 (R123, 5 µM) were evaluated in control and recombinant cells. Additionally, the transepithelial transport of R123 (1 µM) and Vinca alkaloids (5 µM) was evaluated. Except for MDR12677T and MDR11236T/2677T/3435T, cells expressing MDR1 variants displayed intermediate R123 intracellular accumulation (1.5-2‐fold higher) and lower effluxed R123 (10-20% vs. 52%) compared to those expressing MDR1wt. Efflux ratios across MDR1wt expressing cells were significantly larger for R123 (3.95 ± 1.1), Vinblastine (3.75 ± 0.26), and Vincristine (2.8 ± 0.29). Recombinant cells expressing MDR1 variants displayed 0%-22.7% P‐gp activity (∼80%-100% efflux loss). Results suggest that MDR1 polymorphisms at the 1236, 2677, and/or 3435 positions significantly minimize P‐gp functionality in vitro, the extent of which appears to be substrate dependant.