Abstract Body: Background: Coordinated electrical activity is essential for normal cardiac function and depends on regulated ion channel gating. Long QT syndrome (LQTS) is a heart rhythm disorder caused by abnormalities in the electrical activity of the heart, leading to dangerous arrhythmias. Mutations in a potassium channel ancillary subunit KCNE3 (T4A, P39R, R99H) are linked to LQTS. However, the structural mechanisms of these mutations remain unclear. We hypothesize that these mutations disrupt KCNE3 structural dynamics and alter the proper channel gating. This study aims to determine LQTS mutation-induced changes in structural dynamics of KCNE3 in lipid bilayers.
Method: Continuous wave EPR spectroscopy coupled with site directed spin labeling was used to assess the effects of LQTS-linked mutations (T4A, P39R, R99H) on KCNE3 structural dynamics in POPC/POPG lipid bilayers. Residues adjacent to mutation sites were replaced with cysteine and labeled with spin label MTSL. Spectral lineshape analysis of 20 spin labeled constructs quantified side chain mobility and rotational correlation times. These data were compared between wild type and mutant proteins. Uncertainties were determined using duplicate sample preparations and data collections. Additionally, 200 ns all atom molecular dynamics simulations were conducted to evaluate structural stability and dynamic behavior.
Result: EPR spectral lineshape analysis revealed mutation dependent changes in spin-label motion of KCNE3 in lipid bilayers. Compared to wild type, KCNE3 mutants L37C-P39R (1.3 vs 0.9 ns), G38C-P39R (1.8 vs 1.1 ns), P39R-D40C (1.9 vs 1.4 ns) showed increased rotational correlation times (τ₀) (±1 ns), indicating reduced local site specific motion in comparison to wild type. However, N5C-T4A (1.3 vs 2.2 ns), G6C-T4A (1.7 vs 2.6 ns), N98C-R99H (1.8 vs 2.3 ns), V100C-R99H (1.5 vs 1.9 ns), and S101C-R99H (0.9 vs 1.5 ns), showed decreased τ₀ values (±1 ns), showing increased site specific motion. Molecular dynamics simulations further complemented experimental findings, demonstrating mutation-dependent changes in the local motional flexibility.
Conclusion: Our data revealed that LQTS-linked mutations induce changes in site specific KCNE3 dynamics that may disrupt channel modulation.