Molecular mechanisms underlying inhibition of full-length protein expression by interaction, with truncated protein of Cav channel

通过与 Cav 通道截短蛋白相互作用抑制全长蛋白表达的分子机制

• 批准号: 14370017
• 负责人: OKAMURA Yasushi
• 金额: $ 6.78万
• 依托单位: Okazaki National Research Institute
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370017/
• 关键词: ion channel; calcium; voltage-gated channel

Truncated calcium channel subunits have been reported in developing tissue or in human channelopaties. However, functional significance of such truncated versions of proteins remain unknown. We studied molecular mechanisms underlying inhibition of expression of full length proteins by truncated proteins in Xenopus oocyte and BHK-G cells. Two electrode voltage clamp and whole cell patch clamp methods were applied on those cells. When truncated mammalian Cav1.2 subunit was coexpressed with the full length cDNA, remarkable suppression of channel expression was observed. This effect occurred at protein level, since introduction of premature stop codon eliminated this suppression. By Western blot analysis, it was shown that truncated protein is transferred into insoluble membrane fraction. Interestingly, the full length protein was also transferred to this fraction on coexpression with the truncated protein, indicating that misfolding of the truncated protein integrates the full length protein via unknown interactions and degradation or suppression of protein synthesis occurs. We also found that some truncated forms of voltage gated sodium channels show dominant-negative effects, suggesting that inhibitory mechanisms of truncated proteins are conserved among 4-motif type voltage gated channels.

在发育中的组织或人类通道中已经报道了截短的钙通道亚基。然而，这种截短形式的蛋白质的功能意义仍然未知。我们研究了非洲爪蟾卵母细胞和BHK-G细胞中截短蛋白抑制全长蛋白表达的分子机制。采用双电极电压钳和全细胞膜片钳技术对细胞进行记录。当截短的哺乳动物Cav1.2亚基与全长cDNA共表达时，观察到通道表达的显著抑制。这种效应发生在蛋白质水平，因为引入提前终止密码子消除了这种抑制。Western印迹分析表明，截短的蛋白质被转移到不溶性膜组分中。有趣的是，全长蛋白质也被转移到与截短的蛋白质共表达的该级分中，表明截短的蛋白质的错误折叠通过未知的相互作用整合全长蛋白质，并且发生蛋白质合成的降解或抑制。我们还发现，一些截断形式的电压门控钠通道显示显性负效应，这表明截断蛋白的抑制机制是保守的4-基序型电压门控通道。

项目成果

Okamura, Y. et al.: "The ascidian dihydropyridine-resistant calcium channel as the prototype of chordate L-type calcium channel"NeuroSignals. 12. 142-158 (2003)

Okamura, Y. 等人：“海鞘二氢吡啶抗性钙通道作为脊索动物 L 型钙通道的原型”神经信号。

Ebihara, T., Komiya, Y., Izumi-Nakaseko, H., Adachi-Akahane, S., Okabe, S., Okamura, Y.: "Coexpression ofa Cav1.2 protein lacking an N-terminus and the first domain specifically suppresses L-type calcium channel activity."FEBS Lett.. 529. 203 (2002)

Ebihara, T.、Komiya, Y.、Izumi-Nakaseko, H.、Adachi-Akahane, S.、Okabe, S.、Okamura, Y.：“缺少 N 末端和第一个结构域的 Cav1.2 蛋白的共表达

Okamura, Y., Izumi-Nakaseko, H., Nakajo, K., Ohtsuka, Y., Ebihara, T.: "The ascidian dihydropyridine-resistant calcium channel as the prototype of chordite L-type calcium channel."Neurosignals. 12(3). 142-158 (2003)

Okamura, Y.、Izumi-Nakaseko, H.、Nakajo, K.、Ohtsuka, Y.、Ebihara, T.：“海鞘二氢吡啶抗性钙通道作为脊索石 L 型钙通道的原型。”神经信号。

Nakajo, K., Katsuyama, Y., Ono, F., Ohtsuka, Y., Okamura, Y.: "Identification, functional characterization and developmental expression of the ascidian Kv4-class potassium channel."Neurosci.Res.. 45. 59-70 (2003)

Nakajo, K.、Katsuyama, Y.、Ono, F.、Ohtsuka, Y.、Okamura, Y.：“海鞘 Kv4 类钾通道的鉴定、功能特征和发育表达。”Neurosci.Res.. 45。

Izumi-Nakaseko, H. et al.: "DHP-insensitive L-type-like Ca channel of ascidian acquires sensitivity to DHP with single amino acid change is domain III P-reigon."FEBS letters. 549. 67-71 (2003)

Izumi-Nakaseko, H. 等人：“海鞘的 DHP 不敏感 L 型 Ca 通道通过结构域 III P-reigon 的单个氨基酸变化获得了对 DHP 的敏感性。”FEBS 字母。