PDF(Pubmed)
Abstract：
UNASSIGNED: 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.
UNASSIGNED: 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 Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments.
UNASSIGNED: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) 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 Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins.
UNASSIGNED: 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 NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.
UNASSIGNED: 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 Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments.
UNASSIGNED: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) 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 Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins.
UNASSIGNED: 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 NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.
摘要：
■1型Andersen-Tawil综合征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Cys154二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。我们通过重组整体Kir2.1通道结构并破坏其开放状态，评估了Cys122至Cys154二硫键处的人类突变是否会导致Kir2.1通道功能障碍和心律失常。
■我们在ATS1家族中鉴定了Kir2.1功能缺失突变(c.366A>T;p.Cys122Tyr)。为了研究其病理生理意义，我们建立了一个表达Kir2.1C122Y变异体的AAV9介导的心脏特异性小鼠模型.我们采用了多学科的方法，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。
■Kir2.1C122Y小鼠概括了ATS1的心电图特征，与性别无关，包括校正的QT延长，传导缺陷,和增加心律失常的易感性。分离的Kir2.1C122Y心肌细胞显示出显着降低的向内整流K（IK1）和向内Na（INa）电流密度，而与正常运输无关。分子动力学预测，在2000-ns模拟中，C122Y突变会引起构象变化，特征在于Kir2.1和磷脂酰肌醇4,5-二磷酸之间的氢键损失比野生型(WT)更大。因此,磷脂酰肌醇4,5-二磷酸结合袋不稳定，导致与WT相比更低的电导状态。因此，从内到外的贴片夹紧，C122Y突变显著减弱了Kir2.1对增加磷脂酰肌醇4,5-二磷酸浓度的敏感性。此外,Kir2.1C122Y突变导致通道体降解，证明Kir2.1和NaV1.5蛋白的时间不稳定性。
■三维Kir2.1通道结构中的细胞外Cys122-to-Cys154二硫键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NaV1.5通道的功能表达，并最终导致1型Andersen-Tawil综合征的传导障碍和危及生命的心律失常。
■我们在ATS1家族中鉴定了Kir2.1功能缺失突变(c.366A>T;p.Cys122Tyr)。为了研究其病理生理意义，我们建立了一个表达Kir2.1C122Y变异体的AAV9介导的心脏特异性小鼠模型.我们采用了多学科的方法，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。
■Kir2.1C122Y小鼠概括了ATS1的心电图特征，与性别无关，包括校正的QT延长，传导缺陷,和增加心律失常的易感性。分离的Kir2.1C122Y心肌细胞显示出显着降低的向内整流K（IK1）和向内Na（INa）电流密度，而与正常运输无关。分子动力学预测，在2000-ns模拟中，C122Y突变会引起构象变化，特征在于Kir2.1和磷脂酰肌醇4,5-二磷酸之间的氢键损失比野生型(WT)更大。因此,磷脂酰肌醇4,5-二磷酸结合袋不稳定，导致与WT相比更低的电导状态。因此，从内到外的贴片夹紧，C122Y突变显著减弱了Kir2.1对增加磷脂酰肌醇4,5-二磷酸浓度的敏感性。此外,Kir2.1C122Y突变导致通道体降解，证明Kir2.1和NaV1.5蛋白的时间不稳定性。
■三维Kir2.1通道结构中的细胞外Cys122-to-Cys154二硫键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NaV1.5通道的功能表达，并最终导致1型Andersen-Tawil综合征的传导障碍和危及生命的心律失常。
