Roux M J, Martinez-Maza R, Le Goff A, Lopez-Corcuera B, Aragon C, Supplisson S
Laboratoire de Neurobiologie, Unité Mixte de Recherche 8544, Centre National de la Recherche Scientifique, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris cedex 05, France.
J Biol Chem. 2001 May 25;276(21):17699-705. doi: 10.1074/jbc.M009196200. Epub 2001 Mar 14.
The neuronal (GlyT2) and glial (GlyT1) glycine transporters, two members of the Na(+)/Cl(-)-dependent neurotransmitter transporter superfamily, differ by many aspects, such as substrate specificity and Na(+) coupling. We have characterized under voltage clamp their reactivity toward the membrane impermeant sulfhydryl reagent [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET). In Xenopus oocytes expressing GlyT1b, application of MTSET reduced to the same extent the Na(+)-dependent charge movement, the glycine-evoked current, and the glycine uptake, indicating a complete inactivation of the transporters following cysteine modification. In contrast, this compound had no detectable effect on the glycine uptake and the glycine-evoked current of GlyT2a. The sensitivities to MTSET of the two transporters can be permutated by suppressing a cysteine (C62A) in the first extracellular loop (EL1) of GlyT1b and introducing one at the equivalent position in GlyT2a, either by point mutation (A223C) or by swapping the EL1 sequence (GlyT1b-EL1 and GlyT2a-EL1) resulting in AFQ <--> CYR modification. Inactivation by MTSET was five times faster in GlyT2a-A223C than in GlyT2a-EL1 or GlyT1b, suggesting that the arginine in position +2 reduced the cysteine reactivity. Protection assays indicate that EL1 cysteines are less accessible in the presence of all co-transported substrates: Na(+), Cl(-), and glycine. Application of dithioerythritol reverses the inactivation by MTSET of the sensitive transporters. Together, these results indicate that EL1 conformation differs between GlyT1b and GlyT2a and is modified by substrate binding and translocation.
神经元型（GlyT2）和胶质细胞型（GlyT1）甘氨酸转运体是Na⁺/Cl⁻依赖性神经递质转运体超家族的两个成员，在许多方面存在差异，如底物特异性和Na⁺偶联。我们在电压钳制条件下研究了它们对膜不透性巯基试剂[2-(三甲基铵)-乙基]-甲硫基磺酸盐（MTSET）的反应性。在表达GlyT1b的非洲爪蟾卵母细胞中，施加MTSET可同等程度地降低Na⁺依赖性电荷移动、甘氨酸诱发电流和甘氨酸摄取，表明半胱氨酸修饰后转运体完全失活。相比之下，该化合物对GlyT2a的甘氨酸摄取和甘氨酸诱发电流没有可检测到的影响。通过在GlyT1b的第一个细胞外环（EL1）中抑制一个半胱氨酸（C62A），并通过点突变（A223C）或交换EL1序列（GlyT1b-EL1和GlyT2a-EL1）在GlyT2a的等效位置引入一个半胱氨酸，可使两种转运体对MTSET的敏感性发生互换，从而导致AFQ <--> CYR修饰。MTSET对GlyT2a-A223C失活的速度比对GlyT2a-EL1或GlyT1b快五倍，这表明+2位的精氨酸降低了半胱氨酸的反应性。保护试验表明，在所有共转运底物（Na⁺、Cl⁻和甘氨酸）存在的情况下，EL1中的半胱氨酸较难接近。应用二硫苏糖醇可逆转MTSET对敏感转运体的失活作用。总之，这些结果表明GlyT1b和GlyT2a的EL1构象不同，且会因底物结合和转运而发生改变。
