Function and mechanism analysis of short QT syndrome related KCNH2 gene variants

在我们的短QT综合征家族中，我们发现了KCNH 2和SLC 4A 3基因的双重突变。但KCNH 2-H70 Y不影响IKr通道动力学，IKr通道在心肌动作电位中起重要作用。因此，我们的目的是阐明SLC 4A 3突变和QTc间期之间的关系。由于SLC 4A 3基因编码AE 3蛋白，其作为Cl-/HCO 3-交换剂用于维持细胞内pH（pHi），因此我们检查了SLC 4A 3-WT和突变体HEK 293稳定细胞的pHi。结果表明，突变体细胞在生理盐溶液和无Cl-溶液之间转换时，H+-当量通量能力较低，交换速度较慢。因此，SLC 4A 3-突变显示通道动力学功能丧失。我们还研究了总细胞裂解物的AE 3蛋白表达，突变细胞显示与WT细胞正常表达。在此基础上，我们将研究重点放在电生理研究上，以验证SLC 4A 3突变体是否直接参与心脏动作电位的3相和4相。然而，在SLC 4A 3 WT和突变体HEK 293稳定细胞系上记录的IKs、IK 1和Ikr野生型（WT）电流无显著差异。我们将KCNH 2-H70 Y质粒转染稳定表达SLC 4A 3突变体的细胞，IKr通道失活时间常数较WT细胞延长，提示在SLC 4A 3突变体影响的异常pHi环境下，KCNH 2-H70 Y使IKr通道失活动力学减慢，可能导致心肌动作电位缩短，导致短QT综合征。

In our short QT syndrome family, we identified double mutations in KCNH2 and SLC4A3 genes. However, the KCNH2-H70Y did not affect IKr channel kinetics, which play an important role in the cardiac action potential. Therefore, we aimed to elucidate the relationship between a SLC4A3 mutation and QTc interval. Because the SLC4A3 gene encodes AE3 protein, which worked as an Cl-/HCO3- exchanger for maintaining intracellular pH (pHi), we examine the pHi of SLC4A3-WT and mutant HEK293 stable cells. We found that mutant cells showed low ability for the H+-equivalent flux and slow exchange speed when extracellular solution changed between physiological salt solution and Cl- free solution. Therefore, the SLC4A3-mutation showed loss-of-function of the channel kinetics.We also studied the AE3 protein expression of total cell lysate, mutant cells showed normal expression with WT cells. Then, we focus our study on the electrophysiological study to check whether the SLC4A3 mutant directly involved in the phase 3 and 4 of cardiac action potential. However, there is no significant difference on the IKs, IK1 and Ikr wild-type (WT) currents, recorded on SLC4A3 WT and mutant HEK293 stable cell lines. In the following, we transfected KCNH2-H70Y plasmid into cells stably expressing SLC4A3 mutant, the IKr channel deactivation time constants were prolonged compared to the WT cells.In conclusion, under the abnormal pHi environment affected by SLC4A3 mutation, KCNH2-H70Y decelerate the IKr deactivation kinetics, which may shorten the cardiac action potential and lead to short QT syndrome.