Figure 2 |. Diphenhydramine recognition by OCT1.

a, ConSurf⁵⁴ analysis of the OCT1_CS central cavity (left). Residue Y36 in the central cavity shows high variability across OCT1 orthologs in the multiple sequence alignment. Detailed DPH-OCT1 interactions in the binding cavity (right), highlighting interacting residues. b, Residual cold competition block of ¹⁴C-metformin uptake mediated by OCT1_CS. 2.5 h pre-treatment with cold competitor performed at the noted concentration, followed by rapid and extensive oocyte washing in ligand-free buffer immediately before initiation of the uptake assay (10 μM ¹⁴C-metformin in 30 min into OCT1_CS expressing oocytes; data background corrected using the water injected controls, and normalized to none-added control condition; n=3 biologically independent replicates shown along with mean ± s.e.m.). c, Functional evaluation of mutants in the OCT1_CS background (accumulation of 10 μM ¹⁴C-metformin in 1 h into mutant-expressing oocytes; ; n=3 biologically independent replicates shown along with mean ± s.e.m.). d, APBS⁵⁵ surface electrostatic calculation of the OCT1_CS central cavity (see Methods). e, Left, uptake of ³H-MPP⁺ by OCT1_CS-GFP or mutants in the OCT1_CS-GFP background in oocytes (accumulation of 100 nM ³H-MPP⁺ in 1 h into mutant-expressing oocytes; n=3 biologically independent replicates shown along with mean ± s.e.m. Data background corrected using the water injected condition, and normalized to the OCT1_CS-GFP uptake condition). Right, uptake of ³H-MPP⁺ by OCT1_CS-GFP or mutants in the OCT1_CS-GFP background expressed in HEK293T cells (accumulation of 10 nM ³H-MPP⁺ in 40 min into mutant-expressing HEK293T cells; n=3 biologically independent replicates shown along with mean ± s.e.m, data background corrected using the mock-transfected condition and normalized to the OCT1_CS-GFP uptake condition). P-values from an unpaired two-tailed t-tests are shown.