Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, Jülich, Germany.
Departments of Neurology and Neurobiology, UCLA School of Medicine, Los Angeles, USA.
Sci Rep. 2017 Oct 24;7(1):13913. doi: 10.1038/s41598-017-14176-4.
SLC1A3 encodes the glial glutamate transporter hEAAT1, which removes glutamate from the synaptic cleft via stoichiometrically coupled Na-K-H-glutamate transport. In a young man with migraine with aura including hemiplegia, we identified a novel SLC1A3 mutation that predicts the substitution of a conserved threonine by proline at position 387 (T387P) in hEAAT1. To evaluate the functional effects of the novel variant, we expressed the wildtype or mutant hEAAT1 in mammalian cells and performed whole-cell patch clamp, fast substrate application, and biochemical analyses. T387P diminishes hEAAT1 glutamate uptake rates and reduces the number of hEAAT1 in the surface membrane. Whereas hEAAT1 anion currents display normal ligand and voltage dependence in cells internally dialyzed with Na-based solution, no anion currents were observed with internal K. Fast substrate application demonstrated that T387P abolishes K-bound retranslocation. Our finding expands the phenotypic spectrum of genetic variation in SLC1A3 and highlights impaired K binding to hEAAT1 as a novel mechanism of glutamate transport dysfunction in human disease.
SLC1A3 编码胶质谷氨酸转运体 hEAAT1，通过化学计量偶联的 Na-K-H-谷氨酸转运将谷氨酸从突触间隙中去除。在一位伴有偏瘫的偏头痛伴先兆的年轻男性中，我们发现了一种新的 SLC1A3 突变，该突变预测 hEAAT1 中第 387 位保守苏氨酸被脯氨酸取代（T387P）。为了评估新型变异体的功能影响，我们在哺乳动物细胞中表达了野生型或突变型 hEAAT1，并进行了全细胞膜片钳、快速底物应用和生化分析。T387P 降低了 hEAAT1 谷氨酸摄取率，并减少了 hEAAT1 在质膜表面的数量。尽管 hEAAT1 阴离子电流在内部用 Na 基溶液透析的细胞中显示出正常的配体和电压依赖性，但在内部用 K 时未观察到阴离子电流。快速底物应用表明 T387P 消除了 K 结合的重摄取。我们的发现扩展了 SLC1A3 中遗传变异的表型谱，并强调了 hEAAT1 中 K 结合的受损作为人类疾病中谷氨酸转运功能障碍的新机制。
