Pathogenic variants at the N-terminal arginine residue 44 disrupt human GABA transporter 1 function: insights from Drosophila epilepsy models

Arginine 44 (R44) is a highly conserved residue in the human GABA transporter 1 (hGAT-1), a member of the solute carrier 6 (SLC6) family, which plays a critical role in regulating inhibitory neurotransmission in the central nervous system. To elucidate its functional importance, we characterized three epilepsy-associated pathogenic variants - R44Q, R44P and R44W - linked to myoclonic-atonic epilepsy (MAE) and developmental delay. Building on evidence that R44 resides within the N-terminal intracellular gate and is essential for transporter function, we employed biochemical, cellular and organismal models (HEK293 cells and <i>Drosophila melanogaster,</i> respectively) to assess the variants' functional impairments, subcellular localization and trafficking. Particular emphasis was placed on R44Q, a validated variant with a pronounced clinical phenotype. The mutants trafficked to the plasma membrane, but were non-functional and exhibited reduced protein stability. <i>In vivo</i>, R44Q displayed cell-type-specific degradation: in astrocytes, GAT was rapidly cleared via proteasomal degradation, whereas in neurons, it showed lower expression with presynaptic enrichment. Proteasome inhibitors (MG-132, bortezomib) and the HDAC inhibitor trichostatin A (TSA) partially rescued R44Q function. Moreover, R44Q-expressing flies presented heat-induced seizures, which were mitigated by 4-phenylbutyrate (4-PBA) treatment. These findings elucidate the molecular basis of R44-mediated hGAT-1 dysfunction and highlight potential therapeutic avenues for SLC6A1-related neurodevelopmental disorders.