Format

Send to

Choose Destination
Exp Neurol. 2019 Jun 6;320:112973. doi: 10.1016/j.expneurol.2019.112973. [Epub ahead of print]

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

Author information

1
Department of Neurology, Vanderbilt University Medical Center, TN 37232, USA; Department of Neurology, Affiliated Hospital, Nantong University, Nantong, Jiangsu 226001, China.
2
Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou 510260, China.
3
Department of Neurology, Vanderbilt University Medical Center, TN 37232, USA.
4
Department of Electrical Engineering & Computer Science and Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA.
5
Neuroscience Graduate Program, Vanderbilt-Meharry Alliance, Vanderbilt University, TN 37235, USA.
6
Guangzhou Women and Children's Medical Center Guangzhou Children's Hospital, Guangzhou, China.
7
Department of Anesthesiology, Vanderbilt University Department of Anesthesiology, Vanderbilt University, Nashville, TN 37232, USA.
8
Institute of Neuroscience and Department of Neurology of the Second Affiliated Hospital of Guangzhou Medical University, Key Laboratory of Neurogenetics and Channelopathies of Guangdong Province and the Ministry of Education of China, Guangzhou 510260, China. Electronic address: stoneyiwu@163.com.
9
Department of Neurology, Vanderbilt University Medical Center, TN 37232, USA. Electronic address: Jingqiong.kang@vanderbilt.edu.

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.

KEYWORDS:

(3)H GABA uptake; Epilepsy; GABA transporter 1; Lennox-Gastaut syndrome; Mutation; Trafficking

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center