Receptor endocytosis is an important mechanism for regulating the synaptic efficacy of neurotransmitters. There is strong evidence that GABA_A receptor endocytosis is clathrin-dependent; however, this process is not well understood. Here we demonstrate that in HEK 293 cells, endocytosis of GABA_A receptors composed of either α₁β₂γ₂Lor α₁β₂ subunits is blocked by the dominant negative dynamin construct K44A. Furthermore, we identify a dileucine AP2 adaptin-binding motif within the receptor β₂ subunit that is critical for endocytosis. Internalization of GABA_A receptors lacking this motif is dramatically inhibited, and the receptors appear to accumulate on the cell surface. Patch clamp analysis of receptors lacking the dileucine motif show that there is an increase in the peak amplitude of GABA-gated chloride currents compared with wild-type receptors. Additionally, GABA-gated chloride currents in HEK 293 cells expressing wild-type receptors are increased by introduction of a peptide corresponding to the dileucine motif region of the receptor β₂ subunit but not by a control peptide containing alanine substitutions for the dileucine motif. In mouse brain cerebral cortical neurons, the dileucine motif peptide increases GABA-gated chloride currents of native GABA_A receptors. This is the first report to our knowledge that an AP2 adaptin dileucine recognition motif is critical for the endocytosis of ligand-gated ion channels belonging to this superfamily.

Adrenocortical carcinoma (ACC) is a rare but deadly cancer for which few treatments exist. Here, we have undertaken a targeted bioinformatics study of The Cancer Genome Atlas (TCGA) ACC dataset focusing on the 30 genes encoding the γ-aminobutyric acid (GABA) system—an under-studied, evolutionarily-conserved system that is an emerging potential player in cancer progression. Our analysis identified a subset of ACC patients whose tumors expressed a distinct GABA system transcriptome. Transcript levels of ABAT (encoding a key GABA shunt enzyme), were upregulated in over 40% of tumors, and this correlated with several favorable clinical outcomes including patient survival; while enrichment and ontology analysis implicated two cancer-related biological pathways involved in metastasis and immune response. The phenotype associated with ABAT upregulation revealed a potential metabolic heterogeneity among ACC tumors associated with enhanced mitochondrial metabolism. Furthermore, many GABA_A receptor subunit-encoding transcripts were expressed, including two (GABRB2 and GABRD) prognostic for patient survival. Transcripts encoding GABA_B receptor subunits and GABA transporters were also ubiquitously expressed. The GABA system transcriptome of ACC tumors is largely mirrored in the ACC NCI-H295R cell line, suggesting that this cell line may be appropriate for future functional studies investigating the role of the GABA system in ACC cell growth phenotypes and metabolism.