Glycine constitutes a major inhibitory neurotransmitter predominantly in caudal regions of the CNS. The extracellular glycine concentration is regulated synergistically by two high affinity, large capacity transporters GlyT1 and GlyT2. Both proteins are encoded by single genes SLC6A9 and SLC6A5, respectively. Mutations within the SLC6A5 gene encoding for GlyT2 have been demonstrated to be causative for hyperekplexia (OMIM #614618), a complex neuromuscular disease, in humans. In contrast, mutations within the SLC6A9 gene encoding for GlyT1 have been associated with GlyT1 encephalopathy (OMIM #601019), a disease causing severe postnatal respiratory deficiency, muscular hypotonia and arthrogryposis. The consequences of the respective GlyT1 mutations on the function of the transporter protein, however, have not yet been analysed. In this study we present the functional characterisation of three previously published GlyT1 mutations, two mutations predicted to cause truncation of GlyT1 (GlyT1^Q573* and GlyT1^K310F+fs*³¹) and one predicted to cause an amino acid exchange within transmembrane domain 7 of the transporter (GlyT1^S407G), that are associated with GlyT1 encephalopathy. Additionally, the characterization of a novel mutation predicted to cause an amino acid exchange within transmembrane domain 1 (GlyT1^V118M) identified in two fetuses showing increased nuchal translucency and arthrogryposis in routine ultrasound scans is demonstrated. We show that in recombinant systems the two presumably truncating mutations resulted in an intracellular retained GlyT1 protein lacking the intracellular C-terminal domain. In both cases this truncated protein did not show any residual transport activity. The point mutations, hGlyT1^S407G and hGlyT1^V118M, were processed correctly, but showed severely diminished activity, thus constituting a functional knock-out in-vivo. Taken together our data demonstrate that all analysed mutations of GlyT1 that have been identified in GlyT1 encephalopathy patients cause severe impairment of transporter function. This is consistent with the idea that loss of GlyT1 function is indeed causal for the disease phenotype.

甘氨酸是一种主要的抑制性神经递质，主要存在于中枢神经系统的尾部区域。细胞外甘氨酸浓度由两种高亲和力、大容量转运蛋白 GlyT1 和 GlyT2 协同调节。两种蛋白质分别由单基因SLC6A9和SLC6A5编码。SLC6A5内的突变已证明编码 GlyT2 的基因是人类复杂神经肌肉疾病 hyperekplexia (OMIM #614618) 的原因。相比之下，编码 GlyT1 的 SLC6A9 基因内的突变与 GlyT1 脑病 (OMIM #601019) 相关，这种疾病会导致严重的产后呼吸缺陷、肌张力减退和关节弯曲。然而，尚未分析相应 GlyT1 突变对转运蛋白功能的影响。在这项研究中，我们展示了三个先前发表的 GlyT1 突变的功能特征，两个预测会导致 GlyT1 截断的突变（GlyT1 ^Q573 * 和 GlyT1 ^K310F+fs * ³¹) 和预测会导致转运蛋白 (GlyT1 ^S407G )跨膜结构域 7 内的氨基酸交换，这与 GlyT1 脑病有关。此外，还^证实了一种新突变的特征，该突变预计会导致跨膜结构域 1 (GlyT1 ^V118M )内的氨基酸交换，在常规超声扫描中显示出增加的颈部半透明和关节^弯曲。我们表明，在重组系统中，两个可能截断的突变导致细胞内保留的 GlyT1 蛋白缺乏细胞内 C 端结构域。在这两种情况下，这种截短的蛋白质都没有显示任何残留的转运活性。点突变，hGlyT1 ^S407G和 hGlyT1 ^V118M，被正确地处理，但表现出严重削弱活性，从而构成功能敲除在-体内。综上所述，我们的数据表明，在 GlyT1 脑病患者中发现的所有分析的 GlyT1 突变都会导致转运蛋白功能严重受损。这与 GlyT1 功能的丧失确实是疾病表型的原因的观点一致。