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Abstract: Inhibitors of the glycine transporter GlyT-1 are being developed as potential treatments for schizophrenia. Here we report on the use of two novel radioligands, [3H]-SB-733993 and [3H]-GSK931145, for the characterisation of GlyT-1 in both cells and native tissue. Binding was evaluated in membranes either from HEK293 cells expressing recombinant human GlyT-1 (hGlyT-1) or from rat cerebral cortex. Specific binding of both [3H]-SB-733993 and [3H]-GSK931145 to hGlyT-1 HEK293 cell membranes and rat cerebral cortex membranes was saturable and comprised >90% of total binding. Kd and B max values for the two radioligands were fairly similar, with Kd values of 1–2 nM and B max values of around 7000 fmol/mg protein in hGlyT-1 membranes and 3000 fmol/mg protein in rat cortex membranes. Association of [3H]-SB-733993 was faster, with binding reaching equilibrium within 30 min compared with 90 min for [3H]-GSK931145. Dissociation was also much slower for [3H]-GSK931145 than for [3H]-SB-733993, with 50% of specific binding being dissociated by approximately 40 min and 5 min, respectively. Autoradiography studies with [3H]-GSK931145 showed widespread distribution of binding in rat brain, with generally higher binding in caudal compared with rostral areas. Initial studies in human frontal cortex membranes showed clear specific binding of [3H]-GSK931145, though with much lower density (B max 570 fmol/mg protein) and slightly lower affinity (Kd 4.5 nM) compared with rat cortex. A human brain autoradiography study showed higher specific binding in cerebellum compared with frontal cortex. All GlyT-1 inhibitors tested, as well as glycine itself, competed fully for the binding of both [3H]-SB-733993 and [3H]-GSK931145 in both hGlyT-1 and rat cortex membranes. Studies on the effect of varying NaCl concentration showed that [3H]-SB-733993 binding was reduced by >90% in the absence of added Na+ ions, whilst [3H]-GSK931145 binding was unaffected. Glycine produced concentration-dependent decreases in binding affinity of both radioligands without major changes in B max values, suggesting that both [3H]-SB-733993 and [3H]-GSK931145 bind to sites on GlyT-1 that are orthosteric to the site at which glycine itself binds. Overall, these results show that both [3H]-SB-733993 and [3H]-GSK931145 are useful radioligands for studies on GlyT-1 in both cell lines and native tissues, with [3H]-GSK931145 being the radioligand of choice for further studies on GlyT-1 expression and pharmacology. [Copyright &y& Elsevier]