A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function
A missense mutation in SLC6A1 associated with Lennox-Gastaut syndrome impairs GABA transporter 1 protein trafficking and function
复制标题
与 Lennox-Gastaut 综合征相关的 SLC6A1 错义突变会损害 GABA 转运蛋白 1 蛋白的运输和功能
DOI：
10.1016/j.expneurol.2019.112973
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发表时间：
2019-10-01
影响因子：
5.3
通讯作者：
Kang, Jing-Qiong
中科院分区：
文献类型：
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作者：
Cai, Kefu;Wang, Jie;Kang, Jing-Qiong
背景：SLC 6A 1基因突变主要与肌阵挛性失张力性癫痫（MAE）和智力残疾有关。我们确定了一种新的错义突变的Lennox-Gastaut综合征（LGS）的患者，其特征是严重的癫痫发作和发育迟缓。通过H-3 γ-氨基丁酸（GABA）摄取，结构建模，活细胞显微镜，细胞表面生物素化和高通量流式细胞术在神经元和非神经元cells.Results：我们发现了一个杂合错义突变（c700 G到A [pG 234 S）在SLC 6A 1编码GABA转运蛋白1（GAT-1）。结构建模表明，突变使全球蛋白质构象不稳定。通过瞬时表达增强型黄色荧光蛋白（YFP）标记的大鼠GAT-1 eDNAs，我们证明了突变型GAT-1（G234 S）转运蛋白在大鼠皮层神经元和HEK 293 T细胞中的总蛋白表达减少。通过高通量流式细胞术分析和活细胞表面生物素化，我们证明了突变体GAT-1（G234 S）具有降低的细胞表面表达。结论：突变GAT-1（G234 S）导致突变转运蛋白不稳定，导致细胞表面和总蛋白水平降低。除了蛋白质表达减少外，该突变还导致GABA摄取减少，导致GABA清除减少，并改变大脑中的GABA能信号传导。受损的运输和减少GABA摄取功能可以解释癫痫患者的表型。
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 H-3 gamma-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 eDNAs, 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. H-3 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.