Osterbur Badhey Marika L, Bertalovitz Alexander C, McDonald Thomas V
Department of Molecular Pharmacology, Department of Medicine, Division of Cardiology, Albert Einstein College of Medicine, Bronx, New York, USA.
J Cardiovasc Electrophysiol. 2017 Sep;28(9):1070-1082. doi: 10.1111/jce.13259. Epub 2017 Jun 23.
Genetic mutations in KCNH2, which encodes hERG, the alpha subunit of the potassium channel responsible for the I current, cause long QT syndrome (LQTS), an inherited cardiac arrhythmia disorder. Electrophysiology techniques are used to correlate genotype with molecular phenotype to determine which mutations identified in patients diagnosed with LQTS are disease causing, and which are benign. These investigations are usually done using heterologous expression in cell lines, and often, epitope fusion tags are used to enable isolation and identification of the protein of interest.
Here, we demonstrate through electrophysiology techniques and immunohistochemistry, that both N-terminal and C-terminal myc fusion tags may perturb hERG protein channel expression and kinetics of the I current. We also characterize the impact of 2 previously reported inadvertent cDNA variants on hERG channel expression and half-life.
Our results underscore the importance of careful characterization of the impact of epitope fusion tags and of confirming complete sequence accuracy prior to genotype-phenotype studies for ion channel proteins such as hERG.
KCNH2基因发生突变，该基因编码hERG，即负责I电流的钾通道的α亚基，会导致长QT综合征（LQTS），这是一种遗传性心律失常疾病。电生理学技术用于将基因型与分子表型相关联，以确定在被诊断为LQTS的患者中鉴定出的哪些突变是致病的，哪些是良性的。这些研究通常使用细胞系中的异源表达来进行，并且通常会使用表位融合标签来分离和鉴定感兴趣的蛋白质。
在此，我们通过电生理学技术和免疫组织化学证明，N端和C端的myc融合标签均可能干扰hERG蛋白通道的表达以及I电流的动力学。我们还表征了2种先前报道的无意cDNA变体对hERG通道表达和半衰期的影响。
我们的结果强调了在对诸如hERG等离子通道蛋白进行基因型-表型研究之前，仔细表征表位融合标签的影响并确认完整序列准确性的重要性。
