The human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) is responsible for clearing GABA from synapse into neurons and astrocytes.Its mutations are known to impair protein folding and trigger epilepsy.Such folding defects are amenable to rescue by chemical and pharmacological chaperones.Our aim is to elucidate molecular features of two novel disease variants, in HEK293 cells (in vitro) and Drosophila melanogaster (in vivo).Deglycosylation experiments (endoglycosidase H(Endo H)) revealed distinct expression patterns for wild type hGAT-1 and the mutants: ER-resident proteins are core-glycosylated,whereas membrane-bound proteins are mature glycosylated.Two protein bands were observed for wild type,while mutants presented either only core-glycosylated species or some mature-glycosylated bands.Subcellular localization of GATs was examined by confocal microscopy (tracking transporters tagged with YFP,and using CFP-tagged calnexin as ER-marker,trypan blue to delineate plasma membranes).Imaging showed both mutants were trapped in cell interior,co-localizing with calnexin.Conversely, wild type hGAT-1 was correctly targeted to cell surface.Hence,we infer that both variants are partially-to-fully misfolded GATs.Radiotracer GABA uptake studies indicate that both mutations induce absolute loss of transport function.Potential of diverse small molecules (chemical chaperone 4-phenylbutyrate (4-PBA),liothyronine and selective hGAT-1 ligands (tiagabine)) to rescue expression and activity of both mutants is currently underway,but pilot data suggest several compounds are effective (verified by Michaelis-Menten uptake kinetics and Western blotting).Ongoing in vivo experiments in Drosophila melanogaster are well in line with our in vitro data.Collectively,these data ought to impart new mechanistic details and allow for design of effective therapeutic strategies for treatment of hGAT-1-linked syndromes. This work is supported by the Austrian Science Fund (project P36574-B27)