Berberine Induces hERG Channel Deficiency through Trafficking Inhibition
Berberine Induces hERG Channel Deficiency through Trafficking Inhibition
复制标题
DOI：
10.1159/000363034
复制
发表时间：
2014-08
影响因子：
--
通讯作者：
Kai-ping Zhang;Duo Zhi;Ting Huang;Yan Gong;Meng Yan;Chen Liu;Ting Wei;Zeng-xiang Dong;Bao-Xin Li;Baofeng Yang
中科院分区：
文献类型：
--
作者：
Kai-ping Zhang;Duo Zhi;Ting Huang;Yan Gong;Meng Yan;Chen Liu;Ting Wei;Zeng-xiang Dong;Bao-Xin Li;Baofeng Yang
目的：人类乙醚-A-GO-GO相关基因(HERG)编码IKR的α亚基，在动作电位复极过程中起重要作用。HERG通道是各种抗心律失常药物的靶点。黄连素(berberine，BBR)能显著抑制Herg电流，延长动作电位时程。本研究旨在探讨BBR对135 kDa/155 kDa HERG基因表达的长期影响及其机制。方法和结果：用免疫印迹法检测HERG的表达。BBR使成熟的HERG(155 KDa)减少，而内质网定位的HERG(135 KDa)增加。这表明HERG仅限于ER，而BBR中断了渠道贩运。为了确定转运抑制的机制，我们通过蛋白质印迹和免疫沉淀来检测HERG的折叠，通过评估HERG与Hsp90/Hsp70的相互作用。BBR可显著降低Hsp90的表达及其与HERG的相互作用。这些数据表明，BBR减少了通道折叠，从而诱导运输抑制。Western印迹和共聚焦成像进一步检测未折叠蛋白反应(UPR)是否被激活。活化的ATF6是UPR的标志物，被BBR激活。被ATF6激活以协助通道折叠的伴侣蛋白Calnexin和Calreticrin也升高，并与HERG共存。这些数据还表明，普遍定期审议已启动。在BBR治疗后进行免疫沉淀和蛋白质印迹分析，以检测泛素化和降解，这是UPR的共同终点。我们发现内质网限制的HERG在溶酶体和蛋白酶体中被泛素化和降解。结论：BBR可通过抑制孵育24小时后的HERG通道转运而导致HERG通道缺陷。运输抑制激活了UPR，内质网限制的HERG在溶酶体和蛋白酶体中被泛素化和降解。
Aims: The human ether-a-go-go-related gene (hERG) encodes the α subunit of the IKr, which plays an essential role in repolarization of action potentials. hERG channels are targeted by various pro-arrhythmic drugs. Berberine (BBR) was previously found to acutely inhibit hERG currents and prolong action potential duration. The present study aimed to determine long-term effects of BBR on the expression of 135kDa/155kDa hERG and the mechanism. Methods and Results: hERG expression was assessed by western blot. Mature hERG (155 kDa) was reduced, whereas ER-located hERG (135 kDa) was increased by BBR. This indicated that hERG was restricted to the ER and that BBR disrupted channel trafficking. To determine the mechanism of trafficking inhibition, we performed western blot and immunoprecipitation to test folding of hERG by assessing interaction between hERG and Hsp90/Hsp70. Both the expression of Hsp90 and its interaction with hERG were strongly decreased by BBR. These data suggest that BBR reduces channel folding to induce trafficking inhibition. Western blot and confocal imaging were used to further detect whether the unfolded protein response (UPR) was activated. Active ATF6, a marker of the UPR, was activated by BBR. Calnexin and calreticulin, chaperones that are activated by ATF6 to assist channel folding, were also elevated and increasingly colocalized with hERG. These data also demonstrate that the UPR was activated. Immunoprecipitation and western blot assays were performed after BBR treatment to examine ubiquitination and degradation, common endpoints of the UPR. We found that the ER-restricted hERG was ubiquitinized and degraded in the lysosomes and proteasomes. Conclusion: Our study demonstrates that BBR induces hERG channel deficiency by inhibiting channel trafficking after incubation for 24h. Trafficking inhibition activated the UPR, and the ER-restricted hERG was ubiquitinized and degraded in lysosomes and proteasomes.