Mikuni Ikuomi, Torres Carlos G, Bienengraeber Martin W, Kwok Wai-Meng
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Biochim Biophys Acta. 2011 Dec;1810(12):1285-93. doi: 10.1016/j.bbagen.2011.07.018. Epub 2011 Aug 10.
The A341V mutation in the pore-forming KCNQ1 subunit of the slowly activating delayed-rectifier potassium current (IKs) underlies a common form of the long QT syndrome, and is associated with an unusually severe phenotype. However, there is controversy regarding the underlying mechanism responsible for the clinically observed phenotype. We investigated the biophysical characteristics of A341V in a cardiac environment by utilizing a cardiac cell line, and in particular the impact of the KCNE1 β-subunit.
Whole-cell current were recorded from transiently transfected HL-1 cells, a cardiac cell line. Mutant KCNQ1 and KCNE1 were constructed by site-directed mutagenesis.
The A341V mutant resulted in a non-functional channel when expressed alone. When co-expressed with wild type KCNE1, A341V produced a slowly activating current, with a smaller current density, slower rates of activation, and a depolarized shift in its activation curve compared to the wild type KCNQ1+KCNE1. Confocal microscopy confirmed the surface expression of GFP-tagged A341V, suggesting a functionally defective protein. A T58A mutation in KCNE1 abolished functional restoration of A341V. Under heterozygous conditions, the expression of A341V+KCNQ1+KCNE1 reduced but did not abolish the electrophysiological changes observed in A341V+KCNE1. A dominant negative effect of A341V was also observed. Action potential simulations revealed that the A341V mutation is arrhythmogenic.
The KCNE1 β-subunit partially rescued the non-functional A341V mutant, with electrophysiological properties distinct from the wild type IKs.
The severity of the A341V phenotype may be due to a combination of a significant suppression of the IKs with altered biophysical characteristics.
缓慢激活延迟整流钾电流（IKs）的孔形成KCNQ1亚基中的A341V突变是长QT综合征的一种常见形式的基础，并且与异常严重的表型相关。然而，关于导致临床观察到的表型的潜在机制存在争议。我们通过利用一种心脏细胞系，研究了心脏环境中A341V的生物物理特性，特别是KCNE1β亚基的影响。
从瞬时转染的HL-1细胞（一种心脏细胞系）记录全细胞电流。通过定点诱变构建突变型KCNQ1和KCNE1。
单独表达时，A341V突变体导致通道无功能。与野生型KCNE1共表达时，A341V产生缓慢激活的电流，与野生型KCNQ1+KCNE1相比，电流密度较小，激活速率较慢，其激活曲线发生去极化偏移。共聚焦显微镜证实了绿色荧光蛋白标记的A341V的表面表达，表明存在功能缺陷的蛋白质。KCNE1中的T58A突变消除了A341V的功能恢复。在杂合条件下，A341V+KCNQ1+KCNE1的表达减少但并未消除在A341V+KCNE1中观察到的电生理变化。还观察到A341V的显性负效应。动作电位模拟显示A341V突变具有致心律失常性。
KCNE1β亚基部分挽救了无功能的A341V突变体，其电生理特性与野生型IKs不同。
A341V表型的严重程度可能是由于IKs的显著抑制与生物物理特性改变共同作用的结果。
