Extended Data Fig. 10: Functional characterisation of HsOAT1 using 6-carboxyfluorescein.

a, The fluorescent model substrate 6-carboxyfluorescein was used to assess the function of HsOAT1 overexpressed in HeLa cells. The substrate was taken up by the cells over expressing WT HsOAT1 (WT) to a high level compared to cells transfected with the empty vector (Empty). The amount of compound also increased over time and was fully inhibited through the addition of 0.1 mM pemetrexed (+P) to the assay buffer. A truncated version which lacked the last 10 amino acids (Truncation), akin to the construct used for structural determination, did not show any difference in uptake behaviour to WT protein. The C-terminal region of OAT1 had also been previous reported to be non-required for transport⁶⁴. n = 4 independent experiments with error bars showing standard deviation. b, The 6-CF assay shows a similar substrate specificity for OAT1 as reported within the literature²⁰ extending from known good substrate (α-ketoglutarate, p-aminobenzoic acid), to poor substrates (pemetrexed, cefadroxil). All substrates were added at 0.5 mM to the buffer and the data shown is uptake after 8 minutes. n = 6 independent experiments with error bars showing standard deviation. c, Tenofovir modelled into the density observed in site 3 as described in the study (Left). If the ligand is flipped 180 °C (right), the fit to the density and favourable interactions are worse. d and e, Probenecid modelled into the density observed in site 3 as described in the study (Left). If the ligand is flipped 180 °C (right), the fit to the density and favourable interactions are worse.