Cell membranes consist of heterogeneous lipid nanodomains that influence key cellular processes. Using FRET-based fluorescent assays and fluorescence lifetime imaging microscopy (FLIM), we find that the dimension of cholesterol-enriched ordered membrane domains (OMD) varies considerably, depending on specific cell types. Particularly, nociceptor dorsal root ganglion (DRG) neurons exhibit large OMDs. Disruption of OMDs potentiated action potential firing in nociceptor DRG neurons and facilitated the opening of native hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels. This increased neuronal firing is partially due to an increased open probability and altered gating kinetics of HCN channels. The gating effect on HCN channels is likely due to a direct modulation of their voltage sensors by OMDs. In animal models of neuropathic pain, we observe reduced OMD size and a loss of HCN channel localization within OMDs. Additionally, cholesterol supplementation inhibited HCN channels and reduced neuronal hyperexcitability in pain models. These findings suggest that disturbances in lipid nanodomains play a critical role in regulating HCN channels within nociceptor DRG neurons, influencing pain modulation.

细胞膜由影响关键细胞过程的异质脂质纳米结构域组成。使用基于 FRET 的荧光测定和荧光寿命成像显微镜 （FLIM），我们发现富含胆固醇的有序膜结构域 （OMD） 的尺寸差异很大，具体取决于特定的细胞类型。特别是，伤害感受器背根神经节 （DRG） 神经元表现出较大的 OMD。OMD 的破坏增强了伤害感受器 DRG 神经元中的动作电位放电，并促进了天然超极化激活的环核苷酸门控 （HCN） 起搏器通道的打开。这种神经元放电的增加部分是由于 HCN 通道的开放概率增加和门控动力学改变。对 HCN 通道的选通效应可能是由于 OMD 直接调制其电压传感器。在神经性疼痛的动物模型中，我们观察到 OMD 大小减小和 OMD 内 HCN 通道定位的丢失。此外，胆固醇补充剂抑制了疼痛模型中的 HCN 通道并降低了神经元过度兴奋。这些发现表明，脂质纳米结构域的干扰在调节伤害感受器 DRG 神经元内的 HCN 通道中起关键作用，影响疼痛调节。