Org-25543 is a biologically irreversible GlyT2 inhibitor. Glycine-evoked currents in oocytes expressing human GlyT2. (A, C, E) representative reversibility experiments displaying currents induced by three pulses of glycine in the presence of NAGly (A), Org-25543 (C) or ALX-1393 (E). (B, D, F) Concentration–response curves showing the % of glycine-induced current of the second peak (black) or the third peak (white, following washout) as compared with the first glycine current. Org-25543, but not NAGly or ALX-1393 was irreversible in this assay. Each point represents the average response of five to eight oocytes ± SEM.