The dominant negative LQT2 mutation A561V reduces wild-type HERG expression
The dominant negative LQT2 mutation A561V reduces wild-type HERG expression
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DOI：
10.1074/jbc.275.15.11241
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发表时间：
2000-04-14
影响因子：
4.8
通讯作者：
McDonald, TV
中科院分区：
文献类型：
--
作者：
Kagan, A;Yu, ZH;McDonald, TV
HERG（1）K+通道突变是导致显性遗传性长QT综合征（LQT）的一种原因。一些LQT突变对野生型电流表达产生显性负效应。为了研究显性负性行为的机制，我们在哺乳动物细胞中共表达野生型HERG与A561V突变体。不同cDNA比例的转染产生的HERG K+电流密度接近预测的二项分布，其中突变体和野生型亚基共组装在四聚体中，几乎完全占优势。使用C末端myc标记的野生型HERG，我们特别关注突变体对全长野生型HERG蛋白表达的影响。与A561V共表达降低了全长野生型HERG蛋白的丰度，与当前降低相当。野生型蛋白质的减少是由于合成减少和周转增加。促进蛋白质折叠的条件（生长在30 ℃，或在10%甘油）导致从显性效应的部分救援，如26 S蛋白体抑制剂ALLN。因此，对于A561V，显性负效应是由野生型亚基与突变体在生产中非常早期的组装引起的，导致突变体通道的快速识别和蛋白水解的靶向。这些结果确立了蛋白质错误折叠、细胞校正和旁观者参与是LQT 2中显性效应的贡献机制。
HERG(1) K+ channel mutations are responsible for one form of dominantly inherited Long QT syndrome (LQT), Some LQT mutations exert a dominant negative effect on wild-type current expression. To investigate mechanisms of dominant-negative behavior, we co-expressed wild-type HERG with the A561V mutant in mammalian cells. Transfection with various cDNA ratios produced HERG K+ current densities that approached a predicted binomial distribution where mutant and wild-type subunits co-assemble in a tetramer with nearly complete dominance. Using C terminus myc-tagged wild-type HERG we specifically followed the mutant's effect on full-length wild-type HERG protein expression. Co-expression with A561V reduced the abundance of full-length wild-type HERG protein comparable to the current reduction. Reduction of wild-type protein was due to decreased synthesis and increased turnover. Conditions facilitating protein folding (growth at 30 degrees C, or in 10% glycerol) resulted in partial rescue from the dominant effect, as did the 26 S proteosome inhibitor ALLN. Thus, for A561V, dominant negative effects result from assembly of wild-type subunits with mutant very early in production leading to rapid recognition of mutant channels and targeting for proteolysis. These results establish protein misfolding, cellular proofreading, and bystander involvement as contributing mechanisms for dominant effects in LQT2.