Atomic-Level Investigation of KCNJ2 Mutations Associated with Ventricular Arrhythmic Syndrome Phenotypes

Abstract

KCNJ2 encodes the inward rectifying potassium channel (Kir2.1) that creates IK1 which maintains the cardiac resting membrane potential and regulates excitability. Mutations in KCNJ2 have been linked to several clinical phenotypes associated with ventricular arrhythmia and sudden death including Andersen-Tawil syndrome (ATS) related to loss of function mutations and Short QT Syndrome 3 related to gain of function mutations. Detailed structural-functional relationships to explain the arrhythmia phenotypes are understudied and limit the capacity to provide precision medicine. Here, we combine in-depth and complementary computational molecular modeling techniques with functional analysis from three patients with ATS that harbor KCNJ2 mutations R67Q, R218L, and G300D. Whole-cell patch-clamp experiments revealed loss of function in homomeric mutant channels. Full-length Kir2.1 models were developed for structure-based investigation, and mutations were introduced in both open and closed conformations. Site-directed mutagenesis identified altered interaction profiles contributing to structural perturbations. Molecular dynamics simulations assessed the impact of each mutation on overall channel conformation and stability. Principal component analysis and normal mode analysis revealed mutation-specific structural perturbations. These findings extend beyond previous studies, offering atomic-level characterization of mutation-specific perturbations. Our multifaceted approach provides first atomic-level insights into the molecular mechanisms underlying ATS, paving the way for targeted therapeutic strategies. Study Highlights1) Clinical mutation analysis confirmed loss-of-function. 2) Mutation analysis revealed that these clinical mutations dramatically alter the interaction pattern of the mutated residue and subsequently disturbs channel stability. 3) The molecular dynamics based RMSD and RMSF evaluations show that the open conformation state of the channel is more stable comparative to the closed state however, the mutations impact channel conformations regardless of conductance state. 4) The PCA (principal component analysis) and PCA based NM (normal mode) analysis revealed that these clinical KCNJ2 mutations caused significant conformational changes, even distant from the specific residue. 4) This study is the first extensive in silico and experimental analysis of KCNJ2 clinical mutations that start from the arrhythmia phenotype and lead to an in-depth atomic-level investigation. These newly resolved features pave the way towards a better understanding of the molecular disease mechanism and new therapeutic strategies.