Hu Rou-Mu, Tan Bi-Hua, Tester David J, Song Chunhua, He Yang, Dovat Sinisa, Peterson Blaise Z, Ackerman Michael J, Makielski Jonathan C
Department of Cardiology, China Meitan General Hospital, Beijing, China; Department of Medicine, Division of Cardiovascular Medicine, University of Wisconsin, Madison, WI, United States of America.
Department of Medicine, Division of Cardiovascular Medicine, University of Wisconsin, Madison, WI, United States of America; Departments of Pediatrics, and Cellular & Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA, United States of America.
PLoS One. 2015 Apr 29;10(4):e0124921. doi: 10.1371/journal.pone.0124921. eCollection 2015.
SCN5A is a susceptibility gene for type 3 long QT syndrome, Brugada syndrome, and sudden infant death syndrome. INa dysfunction from mutated SCN5A can depend upon the splice variant background in which it is expressed and also upon environmental factors such as acidosis. S1787N was reported previously as a LQT3-associated mutation and has also been observed in 1 of 295 healthy white controls. Here, we determined the in vitro biophysical phenotype of SCN5A-S1787N in an effort to further assess its possible pathogenicity.
We engineered S1787N in the two most common alternatively spliced SCN5A isoforms, the major isoform lacking a glutamine at position 1077 (Q1077del) and the minor isoform containing Q1077, and expressed these two engineered constructs in HEK293 cells for electrophysiological study. Macroscopic voltage-gated INa was measured 24 hours after transfection with standard whole-cell patch clamp techniques. We applied intracellular solutions with pH7.4 or pH6.7. S1787N in the Q1077 background had WT-like INa including peak INa density, activation and inactivation parameters, and late INa amplitude in both pH 7.4 and pH 6.7. However, with S1787N in the Q1077del background, the percentages of INa late/peak were increased by 2.1 fold in pH 7.4 and by 2.9 fold in pH 6.7 when compared to WT.
The LQT3-like biophysical phenotype for S1787N depends on both the SCN5A splice variant and on the intracellular pH. These findings provide further evidence that the splice variant and environmental factors affect the molecular phenotype of cardiac SCN5A-encoded sodium channel (Nav1.5), has implications for the clinical phenotype, and may provide insight into acidosis-induced arrhythmia mechanisms.
SCN5A是3型长QT综合征、Brugada综合征和婴儿猝死综合征的易感基因。SCN5A突变导致的钠电流（INa）功能障碍可能取决于其表达的剪接变体背景，也取决于诸如酸中毒等环境因素。S1787N先前被报道为与LQT3相关的突变，并且在295名健康白人对照中的1名中也观察到了该突变。在此，我们确定了SCN5A - S1787N的体外生物物理表型，以进一步评估其可能的致病性。
我们在两种最常见的选择性剪接SCN5A异构体中构建了S1787N，一种是在第1077位缺乏谷氨酰胺的主要异构体（Q1077del），另一种是含有Q1077的次要异构体，并将这两种构建体在HEK293细胞中表达以进行电生理研究。转染24小时后，用标准的全细胞膜片钳技术测量宏观电压门控INa。我们应用pH7.4或pH6.7的细胞内溶液。在pH7.4和pH6.7条件下，Q1077背景中的S1787N具有类似野生型（WT）的INa，包括峰值INa密度、激活和失活参数以及晚期INa幅度。然而，对于Q1077del背景中的S1787N，与WT相比，pH7.4时INa晚期/峰值的百分比增加了2.1倍，pH6.7时增加了2.9倍。
S1787N的LQT3样生物物理表型取决于SCN5A剪接变体和细胞内pH。这些发现提供了进一步的证据，表明剪接变体和环境因素会影响心脏SCN5A编码的钠通道（Nav1.5）的分子表型，对临床表型有影响，并可能为酸中毒诱导的心律失常机制提供见解。
