Cholesterol Inhibits HCN Channels through Dual Mechanisms in Neuropathic Pain

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. Depletion of membrane cholesterol potentiated HCN channel opening and accelerated activation kinetics, whereas cholesterol supplementation reduced channel opening and slowed activation kinetics. However, the relative contributions of cholesterol that organizes ordered membrane domains (OMDs) versus freely accessible cholesterol pools to HCN channel modulation remain unknown. Using fluorescence lifetime imaging microscopy and Forster resonance energy transfer (FRET), and fluorescence anisotropy techniques, we examined how supplementing cholesterol alters plasma membrane properties and HCN gating in nociceptor DRG neurons. 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. Notably, the slope factor of the HCN conductance- voltage relationship is sensitive to OMD expansion but remains unaffected by changes in free cholesterol. Other gating parameters, including open probability and activation kinetics, were affected by elevating free cholesterol. In a rat model of nerve injury, where DRG neurons exhibit reduced free cholesterol levels and smaller OMDs, HCN channel modulation by cholesterol involves contributions from both OMD expansion and free cholesterol accumulation. In contrast, in naive DRG neurons--characterized by high cholesterol and large OMDs--modulation occurs mostly via increased free cholesterol. These findings deepen our understanding of cholesterols role in modulating ion channels and contributing to neuropathic pain.