Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome

BackgroundAndersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested 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 the open state of the channel. Methods and ResultsWe identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 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 PIP2 concentrations. ConclusionThe extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias. CLINICAL PERSPECTIVEO_ST_ABSNOVELTY AND SIGNIFICANCEC_ST_ABSWhat is known? O_LIAndersen-Tawil Syndrome Type 1 (ATS1) is a rare arrhythmogenic disease caused by loss-of-function mutations in KCNJ2, the gene encoding the strong inward rectifier potassium channel Kir2.1 responsible for IK1. C_LIO_LIExtracellular Cys122 and Cys154 form an intramolecular disulfide bond that is essential for proper Kir2.1 channel folding but not considered vital for channel function. C_LIO_LIReplacement of Cys122 or Cys154 residues in the Kir2.1 channel with either alanine or serine abolished ionic current in Xenopus laevis oocytes. C_LI What new information does this article contribute? O_LIWe generated a mouse model that recapitulates the main cardiac electrical abnormalities of ATS1 patients carrying the C122Y mutation, including prolonged QT interval and life-threatening ventricular arrhythmias. C_LIO_LIWe demonstrate for the first time that a single residue mutation causing a break in the extracellular Cys122-to-Cys154 disulfide-bond leads to Kir2.1 channel dysfunction and arrhythmias in part by reorganizing the overall Kir2.1 channel structure, disrupting PIP2-dependent Kir2.1 channel function and destabilizing the open state of the channel. C_LIO_LIDefects in Kir2.1 energetic stability alter the functional expression of the voltage-gated cardiac sodium channel Nav1.5, one of the main Kir2.1 interactors in the macromolecular channelosome complex, contributing to the arrhythmias. C_LIO_LIThe data support the idea that susceptibility to arrhythmias and SCD in ATS1 are specific to the type and location of the mutation, so that clinical management should be different for each patient. C_LIO_LIAltogether, the results may lead to the identification of new molecular targets in the future design of drugs to treat a human disease that currently has no defined therapy. C_LI