(a) Inhibition of GlyT2 by 1 µM oleoyl L-carnitine. Transporters were WT or with mutations to the vestibule allosteric site (VAS) or adjacent to EL4. The reduced glycine transport currents were normalised to the current elicited by glycine alone. Data represented are means ± SEM with ****p<0.001 following a one way ANOVA test. (b–e) All lipid inhibitors docked to GlyT2 and burrowed into an area between EL4, TM1, TM5, TM7, and TM8 during 100 ns of simulation. (b) The initial docked poses, viewed top down from the extracellular side of GlyT2 (grey surface). Sections of TM1, TM5, and TM7 are cut away in (b–d) to show the docking cavity (transparent surface). (c) The initial docked poses of OLLys (orange sticks), OLTrp (green sticks), OLLeu (blue sticks), and OLSer (magenta sticks) overlaid on GlyT2. (d) The conformations of the lipid inhibitors after 100 ns of unrestrained MD simulation, overlaid on GlyT2 from the OLLys simulation. (e) A close-up view of the inhibitors following 100 ns of simulation, with surrounding TM helices, including those cut away in panels (b–d), shown as a coloured surface. (f–g) Map of key regions in the extracellular allosteric site. (f) 3D arrangement of residues within 4 Å of OLLys (black sticks) following 100 ns of simulation. Residues have side chains shown as sticks, with side chains coloured TM1 (red), TM5 (yellow), TM7 (purple), EL4 (orange), TM8 (green). V523 is a backbone interaction. F428, R439, and L569 are >4 Å but shown for reference. (g) 2D representation, with residues studied via mutagenesis outlined in black.