在哺乳动物细胞中高效表达网状肽门控离子通道。

Michèle Bachmann, Audrey Ortega-Ramírez, Lilia Leisle, Stefan Gründer
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摘要

水螅 Na+ 通道(HyNaCs)是 DEG/ENaC 基因家族的肽门控离子通道,可被水螅神经系统的神经肽直接激活。此前,它们已在爪蟾卵母细胞中成功表征。为了确定它们在哺乳动物细胞中的表达,我们在人类 HEK 293 细胞和猴子 COS-7 细胞中瞬时表达了异构 HyNaC2/3/5。我们发现,使用原生 cDNA 表达 HyNaC2/3/5 的效率很低,而在贴片钳实验中,经过密码子优化的 HyNaC2/3/5 蛋白表达量和电流幅度都有很大提高。我们利用经过密码子优化的通道亚基表达量的提高进行了 Ca2+ 成像,并展示了它们的糖基化模式。总之,我们在哺乳动物细胞系中建立了一种刺胞动物离子通道的高效表达。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Efficient expression of a cnidarian peptide-gated ion channel in mammalian cells.

Hydra Na+ channels (HyNaCs) are peptide-gated ion channels of the DEG/ENaC gene family that are directly activated by neuropeptides of the Hydra nervous system. They have previously been successfully characterized in Xenopus oocytes. To establish their expression in mammalian cells, we transiently expressed heteromeric HyNaC2/3/5 in human HEK 293 and monkey COS-7 cells. We found that the expression of HyNaC2/3/5 using native cDNAs was inefficient and that codon optimization strongly increased protein expression and current amplitude in patch-clamp experiments. We used the improved expression of codon-optimized channel subunits to perform Ca2+ imaging and to demonstrate their glycosylation pattern. In summary, we established efficient expression of a cnidarian ion channel in mammalian cell lines.

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