Ontology highlight
ABSTRACT: Background
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.Methods
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.Results
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).Conclusions
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.
SUBMITTER: Cai K
PROVIDER: S-EPMC6849469 | biostudies-literature | 2019 Oct
REPOSITORIES: biostudies-literature
Cai Kefu K Wang Jie J Eissman Jaclyn J Wang Juexin J Nwosu Gerald G Shen Wangzhen W Liang Hui-Ci HC Li Xiao-Jing XJ Zhu Hai-Xia HX Yi Yong-Hong YH Song Jeffrey J Xu Dong D Delpire Eric E Liao Wei-Ping WP Shi Yi-Wu YW Kang Jing-Qiong JQ
Experimental neurology 20190606
<h4>Background</h4>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.<h4>Methods</h4>Exome Sequencing was performed in an epilepsy patient cohort. The impact of the mutation was evaluated by <sup>3</sup>H γ-aminobutyric acid (GABA) uptake, structural modeling, live cell microscopy, cell sur ...[more]