Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia
Molecular Basis of the Dominant Negative Effect of a Glycine Transporter 2 Mutation Associated with Hyperekplexia
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DOI：
10.1074/jbc.m114.587055
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发表时间：
2015-01-23
影响因子：
4.8
通讯作者：
Lopez-Corcuera, Beatriz
中科院分区：
文献类型：
--
作者：
Arribas-Gonzalez, Esther;de Juan-Sanz, Jaime;Lopez-Corcuera, Beatriz
惊跳症是一种罕见的临床综合征，其特征是对轻微的触觉或听觉刺激的反应过度惊吓。这种神经系统疾病可能对新生儿造成严重后果，引起脑损伤和/或因呼吸暂停发作和心肺功能衰竭而猝死。过度兴奋是由抑制性甘氨酸能神经传递缺陷引起的。编码神经元GlyT 2甘氨酸转运蛋白的人SLC 6A 5基因突变是导致该疾病突触前形式的原因。GlyT 2介导突触甘氨酸再循环，其构成甘氨酸能突触处可释放递质的主要来源。尽管迄今为止检测到的大多数GlyT 2突变都是隐性的，但确实存在影响GlyT 2转运的显性负突变。在这项研究中，我们探索的性质和结构的改变的S512 R突变的GlyT 2。我们分析了其显性负效应，即在内质网（ER）中保留野生型GlyT 2，阻止表面表达。我们发现，精氨酸而不是丝氨酸512的存在下，引起转运蛋白的错误折叠，增强协会的ER-伴侣钙连接蛋白，改变协会与外壳蛋白复合物II组件Sec 24 D，从而阻碍ER退出。S512 R突变体与野生型GlyT 2形成寡聚体，导致其保留在ER中。钙连接蛋白的过表达将野生型GlyT 2从突变体的显性负效应中拯救出来，增加了到达质膜的转运蛋白的量，并抑制了野生型和突变型GlyT 2之间的相互作用。在异源细胞和原代神经元中证明了化学分子伴侣克服疾病突变对野生型转运蛋白的显性负效应的能力。
Hyperekplexia or startle disease is a rare clinical syndrome characterized by an exaggerated startle in response to trivial tactile or acoustic stimuli. This neurological disorder can have serious consequences in neonates, provoking brain damage and/or sudden death due to apnea episodes and cardiorespiratory failure. Hyperekplexia is caused by defective inhibitory glycinergic neurotransmission. Mutations in the human SLC6A5 gene encoding the neuronal GlyT2 glycine transporter are responsible for the presynaptic form of the disease. GlyT2 mediates synaptic glycine recycling, which constitutes the main source of releasable transmitter at glycinergic synapses. Although the majority of GlyT2 mutations detected so far are recessive, a dominant negative mutant that affects GlyT2 trafficking does exist. In this study, we explore the properties and structural alterations of the S512R mutation in GlyT2. We analyze its dominant negative effect that retains wild-type GlyT2 in the endoplasmic reticulum (ER), preventing surface expression. We show that the presence of an arginine rather than serine 512 provoked transporter misfolding, enhanced association to the ER-chaperone calnexin, altered association with the coat-protein complex II component Sec24D, and thereby impeded ER exit. The S512R mutant formed oligomers with wild-type GlyT2 causing its retention in the ER. Overexpression of calnexin rescued wild-type GlyT2 from the dominant negative effect of the mutant, increasing the amount of transporter that reached the plasma membrane and dampening the interaction between the wild-type and mutant GlyT2. The ability of chemical chaperones to overcome the dominant negative effect of the disease mutation on the wild-type transporter was demonstrated in heterologous cells and primary neurons.