Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels.

Jeng CJ; Sun MC; Chen YWTang CY

摘要

Episodic ataxia type 2 (EA2) is an autosomal dominant neurological disorder associated with mutations in the gene encoding pore-forming alpha(1A) subunits of human P/Q-type calcium (Ca(V)2.1) channels. The exact mechanism of how mutant channels cause such clinical EA2 features as cerebellar dysfunctions, however, remains unclear. Our previous functional studies in Xenopus oocytes support the idea that EA2 mutants may exert prominent dominant-negative effects on wild-type Ca(V)2.1 channels. To further pursue the mechanism underlying this dominant-negative effect, we examined the effects of EA2 mutants on the subcellular localization pattern of GFP-tagged wild-type Ca(V)2.1 channels in HEK293T cells. In the presence of EA2 mutants, wild-type channels displayed a significant deficiency in membrane targeting and a concurrent increase in cytoplasm retention. Moreover, the cytoplasmic fraction of wild-type channels co-localized with an endoplasmic reticulum (ER) marker, suggesting that a significant amount of wild-type Ca(V)2.1 channels was trapped in the ER. This EA2 mutant-induced ER retention pattern was reversed by lowering the cell incubation temperature from 37 to 27 degrees C. We also inspected the effects of untagged EA2 mutants on the functional expression of GFP-tagged wild-type Ca(V)2.1 channels in HEK293T cells. Whole-cell current density of wild-type channels was diminished in the presence of EA2 mutants, which was also reversed by 27 degrees C incubation. Finally, biochemical analyses indicated that EA2 mutants did not significantly affect the protein expression level of wild-type channels. Taken together, our data suggest that EA2 mutants induce significant ER retention of their wild-type counterparts, thereby suppressing the functional expression of Ca(V)2.1 channels.

机译：情景性共济失调2型(EA2)是一种常染色体主要神经紊乱有关突变基因编码成孔α(1)人类P / q型钙的子单元(Ca (V) 2.1)通道。突变通道引起如此临床EA2特征然而,随着小脑障碍,仍然存在不清楚。非洲爪蟾蜍卵母细胞支持EA2突变体可能起到显著的显性负作用野生型Ca (V) 2.1通道。这一显性负机制效果,我们检查EA2突变体的影响的亚细胞定位模式GFP-tagged野生型HEK293T Ca (2.1 V)渠道细胞。频道显示明显不足膜定位和并发增加细胞质中保留。的野生型渠道硝唑内质网(ER)标记,建议,大量的野生型Ca (2.1 V)通道被困在ER。mutant-induced ER保留模式正好相反通过降低细胞孵化温度37 27度c。我们也检查效果未加标签的EA2突变体的功能2.1表达GFP-tagged野生型Ca (V)渠道HEK293T细胞。野生型通道密度减少EA2突变体的存在,这也是逆转由27摄氏度孵化。生化分析表明EA2突变体没有显著影响蛋白质表达水平的野生型通道。在一起,我们的数据表明,EA2突变体引起显著的ER保留他们野生型相比,从而抑制Ca (V) 2.1通道的功能表达。

Dominant-negative effects of episodic ataxia type 2 mutations involve disruption of membrane trafficking of human P/Q-type Ca2+ channels.

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