Sequential Membrane Remodeling by Cholesterol Distinctly Modulates HCN Channels in Naïve and Neuropathic DRG Neurons

ABSTRACT Cholesterol, abundantly present in distinct plasma membrane pools, is a critical modulator of ion channel function, including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that regulate the excitability of dorsal root ganglion (DRG) nociceptor neurons. However, the relative contributions of cholesterol that organizes ordered membrane domains (OMDs) versus freely accessible cholesterol pools to HCN channel modulation remain unknown. Using advanced FRET techniques, we examined how supplementing cholesterol alters nociceptor DRG neurons in the context of a rat model of nerve injury. We uncovered a process of sequential, stepwise membrane remodeling: an initial phase with OMD expansion and a rapid rise in free cholesterol, followed by continued accumulation of free cholesterol without further OMD expansion. In injured DRG neurons with low cholesterol and reduced OMD size, both mechanisms contribute, whereas in naïve DRG neurons—characterized by high cholesterol and large OMDs—modulation occurs mostly via increased free cholesterol. These findings deepen our understanding of cholesterol’s role in modulating ion channels and contributing to neuropathic pain.