A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function

• Published in: Experimental neurology Vol. 320; p. 112973
• Main Authors: Cai, Kefu, Wang, Jie, Eissman, Jaclyn, Wang, Juexin, Nwosu, Gerald, Shen, Wangzhen, Liang, Hui-Ci, Li, Xiao-Jing, Zhu, Hai-Xia, Yi, Yong-Hong, Song, Jeffrey, Xu, Dong, Delpire, Eric, Liao, Wei-Ping, Shi, Yi-Wu, Kang, Jing-Qiong
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.10.2019
• Subjects: 3H GABA uptake; Adolescent; Animals; Epilepsy; GABA Plasma Membrane Transport Proteins - chemistry; GABA Plasma Membrane Transport Proteins - genetics; GABA Plasma Membrane Transport Proteins - metabolism; GABA transporter 1; HEK293 Cells; HeLa Cells; Humans; Lennox Gastaut Syndrome - genetics; Lennox-Gastaut syndrome; Male; Mutation; Mutation, Missense; Pedigree; Protein Transport - genetics; Rats; Trafficking; Lennox-Gastaut syndrome; GABA transporter 1; Mutation; Trafficking; Epilepsy; 3H GABA uptake; H GABA uptake
• ISSN: 0014-4886; 1090-2430
• DOI: 10.1016/j.expneurol.2019.112973

Mutations in SLC6A1 have been associated mainly with myoclonic atonic epilepsy (MAE) and intellectual disability. We identified a novel missense mutation in a patient with Lennox-Gastaut syndrome (LGS) characterized by severe seizures and developmental delay. Exome Sequencing was performed in an epilepsy patient cohort. The impact of the mutation was evaluated by 3H γ-aminobutyric acid (GABA) uptake, structural modeling, live cell microscopy, cell surface biotinylation and a high-throughput assay flow cytometry in both neurons and non neuronal cells. We discovered a heterozygous missense mutation (c700G to A [pG234S) in the SLC6A1 encoding GABA transporter 1 (GAT-1). Structural modeling suggests the mutation destabilizes the global protein conformation. With transient expression of enhanced yellow fluorescence protein (YFP) tagged rat GAT-1 cDNAs, we demonstrated that the mutant GAT-1(G234S) transporter had reduced total protein expression in both rat cortical neurons and HEK 293 T cells. With a high-throughput flow cytometry assay and live cell surface biotinylation, we demonstrated that the mutant GAT-1(G234S) had reduced cell surface expression. 3H radioactive labeling GABA uptake assay in HeLa cells indicated a reduced function of the mutant GAT-1(G234S). This mutation caused instability of the mutant transporter protein, which resulted in reduced cell surface and total protein levels. The mutation also caused reduced GABA uptake in addition to reduced protein expression, leading to reduced GABA clearance, and altered GABAergic signaling in the brain. The impaired trafficking and reduced GABA uptake function may explain the epilepsy phenotype in the patient. •GABA transporter 1 G234S mutation is associated with Lennox-Gastaut syndrome (<85 characters).•GABA transporter 1 G234S mutation impaired protein trafficking (<85 characters).•GABA transporter 1 G234S reduced the mutant transporter protein expression (<85 characters).•GABA transporter 1 G234S impaired GABA uptake (<85 characters).•Partial loss of function underlies pathophysiology of GABA transporter 1 G234S (<85 characters).