Wilson Andrew J, Quinn Kathryn V, Graves Fiona M, Bitner-Glindzicz Maria, Tinker Andrew
BHF Laboratories and Department of Medicine, University College London, 5 University Street, London, WC1E 6JJ, UK.
Cardiovasc Res. 2005 Aug 15;67(3):476-86. doi: 10.1016/j.cardiores.2005.04.036.
In the hereditary long QT syndromes the commonest defect is in the K+ channel pore forming subunit, KCNQ1. In this study we investigated the role that abnormal KCNQ1 trafficking has in the pathogenesis of the hereditary long QT syndrome (LQT1).
We introduced nine missense and nonsense mutations occurring in LQT1 into the cDNA encoding KCNQ1 fused in frame to the green fluorescent protein. These mutations occur in syndromes that are inherited in both autosomal dominant and recessive fashions. We used biochemistry, electrophysiology and cell imaging to examine the behaviour of wildtype and mutant channel subunits expressed together with the auxiliary subunit KCNE1 expressed in CHO-K1 and C2C12 cells.
We found that a number of mutations in KCNQ1 are retained in the endoplasmic reticulum and unable to translocate to the plasma membrane. Furthermore, some mutations act in a dominant negative fashion and have the ability to suppress the trafficking of wildtype channel. We use fluorescence resonance energy transfer microscopy to show that this occurs because of direct interaction between the mutant subunit and wildtype channel in the endoplasmic reticulum. Finally, a number of specific and nonspecific pharmacological tools are unable to promote the delivery of these mutants to the plasma membrane.
Our data revealed that channel trafficking may contribute to the pathogenesis of LQT1.
在遗传性长QT综合征中，最常见的缺陷是钾离子通道孔形成亚基KCNQ1。在本研究中，我们调查了KCNQ1异常转运在遗传性长QT综合征（LQT1）发病机制中的作用。
我们将LQT1中出现的9种错义突变和无义突变引入编码与绿色荧光蛋白读框融合的KCNQ1的cDNA中。这些突变发生在以常染色体显性和隐性方式遗传的综合征中。我们使用生物化学、电生理学和细胞成像技术来检测野生型和突变型通道亚基与在CHO-K1和C2C12细胞中表达的辅助亚基KCNE1共同表达时的行为。
我们发现KCNQ1中的一些突变保留在内质网中，无法转运到质膜。此外，一些突变以显性负性方式起作用，并有能力抑制野生型通道的转运。我们使用荧光共振能量转移显微镜来表明这种情况的发生是由于内质网中突变亚基与野生型通道之间的直接相互作用。最后，一些特异性和非特异性药理学工具无法促进这些突变体向质膜的转运。
我们的数据表明通道转运可能在LQT1的发病机制中起作用。
