Extracellular Kir2.1 ^C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome

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-Cys 154 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 Cys 122 -to-Cys 154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS AND RESULTS: We identified a Kir2.1 loss-of-function Cys 122 mutation (c.366 A>T; p.Cys122Tyr) in an Andersen-Tawil syndrome type 1 family. We generated a cardiac-specific mouse model expressing the Kir2.1 C122Y that recapitulated the abnormal ECG features of Andersen-Tawil syndrome type 1 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 to and localization at the sarcolemma and the sarcoplasmic reticulum. However, 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 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 V 1.5 proteins. CONCLUSIONS: The extracellular Cys 122 -to-Cys 154 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 V 1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.