The interaction between the first transmembrane domain and the thumb of ASIC1a is critical for its N-glycosylation and trafficking.
The interaction between the first transmembrane domain and the thumb of ASIC1a is critical for its N-glycosylation and trafficking.
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DOI：
10.1371/journal.pone.0026909
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Zha XM
中科院分区：
文献类型：
--
作者：
Jing L;Jiang YQ;Jiang Q;Wang B;Chu XP;Zha XM
酸敏感离子通道 1a (ASIC1a) 是大脑中的主要质子受体，可导致多种疾病，包括中风、癫痫和多发性硬化症。因此，更好地了解其生物发生将为了解 ASIC1a 在疾病中的调节提供重要的见解。有趣的是，ASIC1a 包含一个大但组织良好的胞外域，这表明细胞外侧域-域相互作用的正确形成是 ASIC1a 成熟和运输的关键调节步骤的假设。我们通过关注第一个跨膜结构域 (TM1) 和 ASIC1a 拇指之间的相互作用来测试这一假设，这种相互作用已知在通道门控中至关重要。我们对小鼠 ASIC1a 中参与 TM1-拇指相互作用的两个关键残基 Tyr71 和 Trp287 进行了突变，发现 Y71G 和 W287G 均降低了 ASIC1a 的突触靶向和表面表达。这些缺陷可能是由于折叠方式改变造成的。两种突变体都表现出对胰蛋白酶裂解的抵抗力增强，表明构象发生了变化。此外，两种突变体都缺乏 N 连接聚糖在高尔基体中晚期的成熟。这些数据表明，破坏 TM1 和拇指之间的相互作用会改变 ASIC1a 折叠，阻碍其糖基化并减少其运输。此外，降低培养温度（一种促进蛋白质折叠的常用方法）可增加 ASIC1a 糖基化、表面表达、电流密度并减慢脱敏速度。这些结果表明细胞外胞外域的正确折叠在 ASIC1a 生物发生和功能中起着至关重要的作用。
Acid-sensing ion channel-1a (ASIC1a), the primary proton receptor in the brain, contributes to multiple diseases including stroke, epilepsy and multiple sclerosis. Thus, a better understanding of its biogenesis will provide important insights into the regulation of ASIC1a in diseases. Interestingly, ASIC1a contains a large, yet well organized ectodomain, which suggests the hypothesis that correct formation of domain-domain interactions at the extracellular side is a key regulatory step for ASIC1a maturation and trafficking. We tested this hypothesis here by focusing on the interaction between the first transmembrane domain (TM1) and the thumb of ASIC1a, an interaction known to be critical in channel gating. We mutated Tyr71 and Trp287, two key residues involved in the TM1-thumb interaction in mouse ASIC1a, and found that both Y71G and W287G decreased synaptic targeting and surface expression of ASIC1a. These defects were likely due to altered folding; both mutants showed increased resistance to tryptic cleavage, suggesting a change in conformation. Moreover, both mutants lacked the maturation of N-linked glycans through mid to late Golgi. These data suggest that disrupting the interaction between TM1 and thumb alters ASIC1a folding, impedes its glycosylation and reduces its trafficking. Moreover, reducing the culture temperature, an approach commonly used to facilitate protein folding, increased ASIC1a glycosylation, surface expression, current density and slowed the rate of desensitization. These results suggest that correct folding of extracellular ectodomain plays a critical role in ASIC1a biogenesis and function.