Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1100 South Grand Blvd., St. Louis, MO, 63104, USA.
Nat Commun. 2023 Oct 18;14(1):6595. doi: 10.1038/s41467-023-42363-7.
Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.
离子通道在以动态脂质分隔为特征的膜环境中发挥作用。关于电压门控离子通道在不同膜隔室中的跨膜电位的响应，人们的了解有限。通过荧光寿命成像显微镜 (FLIM) 和Förster 共振能量转移 (FRET)，我们可视化了超极化激活环核苷酸门控 (HCN) 通道在膜域中的定位。与 HCN1 相比，HCN4 更倾向于整合到有序的脂质域中。为了研究 HCN 通道 S4 螺旋电压传感器的构象变化，我们使用双终止密码子抑制来掺入不同的非规范氨基酸、正交点击化学进行位点特异性荧光标记和过渡金属 FLIM-FRET。值得注意的是，在破坏膜隔室后，在 HCN 通道的 VSD 位点之间观察到改变的 FRET 水平。我们提出，电压传感器的重排直接受膜脂质域的影响，这可以解释在胆固醇缺乏、无序脂质域中定位的起搏 HCN 通道活性增加，导致膜过度兴奋和疾病。
