Extracellular cysteine disulfide bond break at Cys122 disrupts PIP₂-dependent Kir2.1 channel function and leads to...

Andersen-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.

We 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 Kir2.1^C122Y mutation. Kir2.1^C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1^C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K⁺ (I_K1) and inward Na⁺ (I_Na) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1^C122Y 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 Kir2.1^C122Y 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.

The 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.