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 naïve 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.