Abstract Body: Background:
The inward rectifier potassium channel Kir2.1 (KCNJ2) is essential for maintaining the cardiac resting membrane potential and supporting phase 3 repolarization. Channel activity is tightly regulated by phosphatidylinositol 4,5 bisphosphate (PIP2), and impaired PIP2 binding contributes to channel dysfunction and inherited arrhythmias, including Andersen Tawil syndrome (ATS). However, the structural and temporal organization of the Kir2.1 PIP2 interaction interface remains incompletely defined.
Objective:
To identify residues critical for PIP2 binding and determine how ATS associated KCNJ2 mutations alter lipid coordination and channel regulation.
Methods:
Long timescale molecular dynamics (MD) simulations (580 ns) were performed on wild type (WT) Kir2.1 and ATS associated variants (R67Q, R218L, and G300D) to characterize PIP2 interactions. Time resolved analyses identified persistent and transient salt bridge and hydrogen bond interactions between Kir2.1 residues and PIP2. To complement these studies, live cell NanoBRET assays were conducted using NanoLuc tagged Kir2.1 and a HaloTag based PIP2 carrier system. Ongoing BRET experiments using a fluorescent lipid probe (BODIPY TMR PIP2) are being performed across a panel of 23 clinical and engineered mutations. Statistical analysis was performed using one way ANOVA with Dunnett’s multiple comparisons test (P<0.05).
Results:
MD simulations revealed a dynamic, conformation dependent PIP2 binding cloud spanning the slide helix, cytoplasmic domain, and membrane interface, consistent with a distributed and hierarchical lipid binding network. Key residues, including K64, R67, R80, K188, R218, and K219, exhibited persistent interactions with PIP2. Among these, R67 and R218 emerged as critical determinants of direct PIP2 binding. In contrast, G300D did not show substantial disruption of PIP2 interactions in MD simulations. Consistent with these findings, NanoBRET assays demonstrated a significant reduction in PIP2 interaction for R67Q and R218L compared with WT, whereas G300D showed a partial, non significant decrease.
Conclusion:
These findings define a distributed and dynamic PIP2 interaction network in Kir2.1. ATS associated mutations within this binding cloud impair PIP2 binding and disrupt channel function, providing mechanistic insight into mutation specific dysfunction. This integrated framework establishes a foundation for targeted therapeutic strategies in KCNJ2 associated arrhythmias.