Acid-sensing ion channels (ASICs) are widely expressed in the nervous system. The intracellular C terminus of ASIC1a has many sites involved in regulating its expression and the opening mechanism, but the role of the intracellular N-terminal domain is poorly understood. Here, we explored the correlation of ASIC1a intracellular N terminus with membrane expression and gate opening. We modified the N-terminal structure of ASICs by deletion/truncation/mutation strategies and transfected the recombinant plasmids into CHO cells. Protein expression was analyzed with immunofluorescence, Western blots, and patch-clamp experiments. Deleting the entire N terminus decreased the membrane expression of channel proteins, and ion channel opening was lost. Deleting sections of the N terminus also decreased membrane expression and suggested that all areas were significant, with no single or group of amino acid residues playing a decisive role in regulating ASIC1a membrane expression. In terms of gate opening, five amino acid (AA) residues from AA 16 to AA 20 participated in gate opening, and isoleucine at AA 18 was the most important. The whole N terminus of ASICs participates in the membrane expression of ASIC1a, and five amino acid residues (AA 16–20) are involved in the gate opening mechanism.

中文翻译：

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酸敏感离子通道 1a 的细胞内 N 端结构域参与通道开放和膜表达

酸敏感离子通道 (ASIC) 在神经系统中广泛表达。ASIC1a 的细胞内 C 末端有许多位点参与调节其表达和开放机制，但细胞内 N 末端结构域的作用知之甚少。在这里，我们探讨了 ASIC1a 细胞内 N 末端与膜表达和门打开的相关性。我们通过删除/截断/突变策略修改了 ASIC 的 N 端结构，并将重组质粒转染到 CHO 细胞中。用免疫荧光、蛋白质印迹和膜片钳实验分析蛋白质表达。删除整个 N 末端会降低通道蛋白的膜表达，并且离子通道开放丢失。删除 N 末端的部分也降低了膜表达，并表明所有区域都很重要，没有单个或一组氨基酸残基在调节 ASIC1a 膜表达中起决定性作用。在门打开方面，AA 16 到 AA 20 的 5 个氨基酸 (AA) 残基参与了门打开，其中 AA 18 的异亮氨酸是最重要的。ASICs 的整个 N 末端参与了 ASIC1a 的膜表达，五个氨基酸残基（AA 16-20）参与了开门机制。