Source data for paper "Voltage Sensor Conformations Induced by LQTS-associated Mutations in hERG Potassium Channels"

In this study, we employed a combination of techniques including nonsense suppression-mediated incorporation of noncanonical amino acids for site-specific fluorescence labeling, FLIM-based transition metal Förster resonance energy transfer (FLIM-tmFRET), and MD simulations to examine the impact of a LQTS-associated mutation on conformational rearrangements of hERG voltage sensors in transfected cell lines. We applied a dual stop-codon suppression strategy relying on a small fluorescent noncanonical amino acid to discern and dissect the conformational heterogeneity of voltage sensors by analyzing the frequency-domain phasor plot function of FLIM-tmFRET. This methodology has significant potential for broad applications, offering an effective approach to quantitatively assess the movement, states, dynamics, and energetics of voltage sensors in ion channels.

本研究结合多种技术手段，包括无义抑制介导的非天然氨基酸（noncanonical amino acids）定点荧光标记技术、基于荧光寿命成像显微镜（Fluorescence Lifetime Imaging Microscopy, FLIM）的过渡金属福斯特共振能量转移（FLIM-tmFRET）技术，以及分子动力学（Molecular Dynamics, MD）模拟，以探究长QT综合征（Long QT Syndrome, LQTS）相关突变对转染细胞系中人类ether-à-go-go相关基因（human Ether-à-go-go Related Gene, hERG）电压传感器构象重排的影响。本研究采用依赖小型荧光非天然氨基酸的双终止密码子抑制策略，通过分析FLIM-tmFRET的频域相图函数，辨别并解析电压传感器的构象异质性。该方法具备广泛应用的巨大潜力，可为定量评估离子通道中电压传感器的运动、状态、动力学及能量学特征提供有效途径。