Extracellular Kir2.1
C122Y
Mutant Upsets Kir2.1-PIP
2
Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome
Authors: Cruz Francisco M. , Macías Álvaro , Moreno-Manuel Ana I. , Gutiérrez Lilian K. , Vera-Pedrosa María Linarejos , Martínez-Carrascoso Isabel , Sánchez Pérez Patricia , Ruiz Robles Juan Manuel , Bermúdez-Jiménez Francisco J. , Díaz-Agustín Aitor , Martínez de Benito Fernando , Arias-Santiago Salvador , Braza-Boils Aitana , Martín-Martínez Mercedes , Gutierrez-Rodríguez Marta , Bernal Juan A. , Zorio Esther , Jiménez-Jaimez Juan , Jalife José 
Published: March 2024

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:

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
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.