Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota; Division of Heart Rhythm Services, Department of Cardiovascular Medicine, Mayo Clinic, Rochester, Minnesota.
Department of Pediatrics, University of Missouri, Kansas City, Missouri.
Heart Rhythm. 2019 Feb;16(2):270-278. doi: 10.1016/j.hrthm.2018.08.030. Epub 2018 Aug 29.
Gain-of-function variants in the CACNA1C-encoded L-type calcium channel (LTCC, Ca1.2) cause type 8 long QT syndrome (LQT8). The pore region contains highly conserved glutamic acid (E) residues that collectively form the LTCC's selectivity filter. Here, we identified and characterized a pore-localizing missense variant, E1115K, that yielded a novel perturbation in the LTCC.
The purpose of this study was to determine whether CACNA1C-E1115K alters the LTCC's selectivity and is the substrate for the patient's LQTS.
The proband was a 14-year-old male with idiopathic QT prolongation and bradycardia. Genetic testing revealed a missense variant, CACNA1C-E1115K. The whole-cell patch clamp technique was used to measure CACNA1C-WT and -E1115K currents when heterologously expressed in TSA201 cells.
The CACNA1C-E1115K channel exhibited no inward calcium current. Instead, robust cardiac transient outward potassium current (I)-like outward currents that were blocked significantly by nifedipine were measured when 2 mM/0.1 mM extracellular/intracellular CaCl2 or 4 mM/141 mM extracellular/intracellular KCl was applied. Furthermore, when 140 mM extracellular NaCl was applied, the CACNA1C-E1115K channel revealed both robust inward persistent Na currents with slower inactivation and outward currents, which were also nifedipine sensitive. In contrast, CACNA1C-WT revealed only a small inward persistent Na current without a robust outward current.
This CACNA1C-E1115K variant destroyed the LTCC's calcium selectivity and instead converted the mutant channel into a channel with a marked increase in sodium-mediated inward currents and potassium-mediated outward currents. Despite the anticipated 50% reduction in LTCC, the creation of a new population of channels with accentuated inward and outward currents represents the likely pathogenic substrates for the patient's LQTS and arrhythmia phenotype.
CACNA1C 编码的 L 型钙通道（LTCC，Ca1.2）中的功能获得性变异导致 8 型长 QT 综合征（LQT8）。孔区含有高度保守的谷氨酸（E）残基，这些残基共同构成 LTCC 的选择性过滤器。在这里，我们鉴定并表征了一个位于孔内的错义变体 E1115K，该变体导致 LTCC 出现新的扰动。
本研究的目的是确定 CACNA1C-E1115K 是否改变 LTCC 的选择性，是否是患者 LQTS 的底物。
先证者为一名 14 岁男性，患有特发性 QT 延长和心动过缓。基因检测显示存在错义变异 CACNA1C-E1115K。当异源表达于 TSA201 细胞时，使用全细胞膜片钳技术测量 CACNA1C-WT 和 -E1115K 电流。
CACNA1C-E1115K 通道没有内向钙电流。相反，当应用 2 mM/0.1 mM 细胞外/细胞内 CaCl2 或 4 mM/141 mM 细胞外/细胞内 KCl 时，测量到强烈的心脏瞬态外向钾电流（I）样外向电流，该电流被硝苯地平显著阻断。此外，当应用 140 mM 细胞外 NaCl 时，CACNA1C-E1115K 通道显示出具有较慢失活的强烈内向持续钠电流和外向电流，这些电流也对硝苯地平敏感。相比之下，CACNA1C-WT 仅显示出小的内向持续钠电流，而没有强烈的外向电流。
这种 CACNA1C-E1115K 变体破坏了 LTCC 的钙选择性，而是将突变通道转换为具有明显增加的钠介导内向电流和钾介导外向电流的通道。尽管 LTCC 预计减少 50%，但创建具有强调内向和外向电流的新通道群体代表了患者 LQTS 和心律失常表型的可能致病底物。
