BACKGROUND:Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K⁺ channel Kir2.1. The extracellular Cys (cysteine)₁₂₂-to-Cys₁₅₄ disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys₁₂₂-to-Cys₁₅₄ disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state.METHODS:We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1^C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments.RESULTS:Kir2.1^C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1^C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (I_K1) and inward Na+ (I_Na) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate–binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1^C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and Na_V1.5 proteins.CONCLUSIONS:The extracellular Cys₁₂₂-to-Cys₁₅₄ disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate–dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the Na_V1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

中文翻译：

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细胞外 Kir2.1C122Y 突变体扰乱 Kir2.1-PIP2 键并导致 Andersen-Tawil 综合征的心律失常

背景：Andersen-Tawil 综合征 1 型是一种罕见的遗传性疾病，由编码强内向整流 K ⁺通道 Kir2.1 的基因突变引起。通道结构中的细胞外 Cys（半胱氨酸）₁₂₂至 Cys ₁₅₄二硫键对于正确折叠至关重要，但与膜上的正确通道功能无关。我们通过重组整个 Kir2.1 通道结构并破坏其开放状态的稳定性，评估了 Cys ₁₂₂至 Cys ₁₅₄二硫桥的人类突变是否会导致 Kir2.1 通道功能障碍和心律失常。方法：我们确定了 Kir2.1 丢失ATS1 家族中的功能突变（c.366 A>T；p.Cys122Tyr）。为了研究其病理生理学意义，我们构建了表达 Kir2.1 ^C122Y变体的 AAV9 介导的心脏特异性小鼠模型。我们采用多学科方法，整合膜片钳和心内刺激、分子生物学技术、分子动力学和生物发光共振能量转移实验。结果：Kir2.1 ^C122Y小鼠重现了独立于性别的 ATS1 心电图特征，包括校正的 QT 延长、传导缺陷，并增加心律失常的易感性。分离的 Kir2.1 ^C122Y心肌细胞显示内向整流 K+ (I _K1 ) 和内向 Na+ (I _Na ) 电流密度显着降低，与正常运输无关。分子动力学预测，C122Y 突变在 2000 纳秒模拟过程中引发构象变化，其特征是与野生型 (WT) 相比，Kir2.1 和磷脂酰肌醇 4,5-二磷酸之间的氢键损失更大。因此，磷脂酰肌醇 4,5-二磷酸结合袋不稳定，导致与 WT 相比电导状态较低。因此，在由内而外的膜片钳作用下，C122Y 突变显着削弱了 Kir2.1 对增加的磷脂酰肌醇 4,5-二磷酸浓度的敏感性。此外，Kir2.1 ^C122Y突变导致通道体降解，证明了 Kir2.1 和 Na _V 1.5 蛋白的时间不稳定性。结论：三维 Kir2.1 通道结构中的胞外 Cys ₁₂₂ -to-Cys ₁₅₄二硫键是对于通道功能至关重要。我们证明，破坏胞外域中的二硫键会破坏磷脂酰肌醇 4,5-二磷酸依赖性调节，导致通道功能障碍和 Kir2.1 能量稳定性缺陷。该突变还改变了 Na _{V的功能表达}1.5 通道，最终导致传导障碍和危及生命的心律失常，这是 1 型 Andersen-Tawil 综合征的特征。