The divergent roles of tryptophans W354 and W217 in OCT1 transport: Similar localization, distinct functions

Hepatic organic cation transporter 1 (OCT1, SLC22A1) has a broad spectrum of structurally diverse substrates, including drugs like morphine, metformin, sumatriptan, and fenoterol. Here, we leveraged existing cryo-EM data to identify amino acids that constrain the binding pocket toward the intracellular lumen and performed functional analyses to determine their roles in OCT1 transport, in general, and in substrate specificity, in particular. We mutated W217, W354, and I446 and analyzed their effects on the transport of 27 structurally highly diverse substrates in HEK293 cells. The W354A mutant resulted in a complete loss of function for all tested substrates, whereas only the W354Y mutant partially retained OCT1 activity. Molecular dynamics simulations and functional analyses suggest that W354 affects OCT1 transport by forming a hydrogen bond with N453 in transmembrane helix 10, thereby stabilizing OCT1 in its outward open conformation. W217A showed strongly substrate-dependent effects, with the aromatic ring of tryptophan being the key property affecting transport. Smaller, less lipophilic substrates were more strongly affected. The substrate-specific effects of the W217A mutant correlated significantly with those of the F244A mutant. The double mutant W217A/F244A strongly reduced uptake of all tested substrates, indicating a compensatory mechanism between these two amino acids. I446 showed only marginal effects on OCT1 transport. Our data suggest that W354 is crucial for OCT1 function by mediating the switch from an inward-occluded to inward-open conformation but is not directly involved in substrate interaction. In contrast, W217, along with the previously known F244, contributes to the substrate specificity of OCT1.