Cholesterol Inhibits HCN Channels through Dual Mechanisms in Neuropathic Pain
Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate the excitability of dorsal root ganglion (DRG) neurons, particularly in the context of neuropathic pain. Cholesterol, a major component of lipid-ordered membrane domains (OMDs), has recently been identified as a critical modulator of HCN channel function. Using FLIM-FRET-based OMD probes and a fluorescent cholesterol sensor GRAM-W, we investigated the effects of cholesterol supplementation on nociceptor DRG neurons from a rat model of spared nerve injury (SNI). We developed a method to distinguish at least two phases of membrane remodeling during cholesterol enrichment: an initial phase marked by OMD expansion and increased accessible cholesterol, followed by a second phase with continued cholesterol accumulation without further OMD expansion. These cholesterol dynamics were further validated through changes in fluorescence anisotropy and homo-FRET measurements of GRAM-W. Temporal analysis of cholesterol enrichment revealed two mechanisms of HCN channel modulation: through expansion of OMDs and elevation of free cholesterol. In SNI DRG neurons with low cholesterol and small OMDs, both mechanisms are active, while in naive DRG neurons--characterized by high cholesterol and large OMDs--modulation occurs only via increased free cholesterol. These findings deepen our understanding of cholesterol's role in modulating ion channels and contributing to neuropathic pain.