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-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.
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 Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments.
RESULTS
Kir2.1C122Y 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.
CONCLUSIONS
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.The authors thank the Centro Nacional de Investigaciones Cardiovasculares (CNIC)
Viral Vectors Unit for producing the adeno-associated virus serotype 9. Confocal experiments were conducted at the CNIC Microscopy and Dynamic Imaging Unit. The
authors thank the CNIC Bioinformatics Unit for generating the in silico homology
modeling simulations, F-function analysis, and helpful discussions. The authors also
thank the Centro de Supercomputación de Galicia for the use of the Finis Terrae III
supercomputer to perform molecular dynamics studies. The CNIC was supported
by the Instituto de Salud Carlos III, the Ministerio de Ciencia, Innovación y Universidades, and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence
(grant CEX2020-001041-S funded by MICIU/AEI/10.13039/501100011033).
This work was supported by the National heart, Lung and Blood Institute under
National Institutes of Health (NIH) grant R01HL163943; the La Caixa Banking
Foundation project code HR18-00304 (grant LCF/PR/HR19/52160013); grants
PI-FIS-2020, PI20/01220, PI-FIS-2023, and PI23/01039 from the Instituto de Salud Carlos III and cofunded by the Fondo Europeo de Desarrollo Regional
(FEDER) and the European Union, respectively; grants PID2020-116935RB-I00
and BFU2016-75144-R funded by MICIU/AEI/10.13039/501100011033; the
Fundación La Marató de TV3 (736/C/2020) amb el suport de la Fundació La Marató
de TV3; the CIBER (Centro de Investigación Biomédica en Red) de Enfermedades
Cardiovasculares (grant CB16/11/00458); the European Union’s Horizon 2020
grant agreement GA-965286; and the Program S2022/BMD7229-CM ARCADIACM funded by the Comunidad de Madrid to J. Jalife; grant PID2021-126423OB-C22
(to M. Martín-Martínez) funded by MICIU/AEI/10.13039/501100011033; and European Regional Development Fund (ERDF) grant PID2022-137214OB-C22 (to
M. Gutierrez-Rodríguez) funded by MICIU/AEI/10.13039/501100011033. The
imaging studies were performed in the TRIMA@CNIC (Infraestructura de Imagen
Traslacional Avanzada del CNIC) node of the ICTS ReDIB (Infraestructuras Científicas y Técnicas Singulares: Red Distribuida de Imagen Biomédica) grant ICTS-2018-
04-CNIC-16 funded by MICIU/AEI/10.13039/501100011033 and ERDF, and
project EQC2018-005070-P funded by MICIU/AEI/10.13039/501100011033
and FEDER. A.I. Moreno-Manuel holds an formación profesional universitaria (FPU)
contract (FPU20/01569) from the Ministerio de Universidades. J.M. Ruiz Robles
holds an FPU contract (FPU22/03253) from the Ministerio de Universidades.
L.K. Gutiérrez holds an FPI contract (PRE2018-083530) from the Ministerio de
Economía y Competitividad de España cofunded by the Fondo Social Europeo, attached to project SEV-2015-0505-18-2. I. Martínez-Carrascoso holds a PFIS (Contratos predoctorales de formación en investigación en salud) contract (FI21/00243)
funded by Instituto de Salud Carlos III and the Fondo Social Europeo Plus cofunded
by the European Union. M.L. Vera-Pedrosa held contract PEJD-2019-PRE/BMD15982 funded by the Consejería de Educación e Investigación de la Comunidad de
Madrid y Fondo Social Europeo.S
