# Sequential membrane remodeling by cholesterol distinctly modulates HCN channels in naïve and neuropathic DRG neurons

## TL;DR

Cholesterol in cell membranes affects pain-sensing neurons by changing how HCN channels work, with different effects in normal and injured nerve cells.

## Contribution

The study reveals how distinct cholesterol pools modulate HCN channels in naïve and neuropathic DRG neurons through sequential membrane remodeling.

## Key findings

- OMD expansion affects the slope factor of HCN G-V relationship but not open probability or activation kinetics.
- Free cholesterol influences HCN channel open probability and activation kinetics.
- Neuropathic DRG neurons show reduced free cholesterol and smaller OMDs compared to naïve neurons.

## Abstract

Cholesterol in cell membranes plays a key role in controlling how pain-sensing neurons respond. Our study shows that distinct cholesterol pools—OMD-associated and freely accessible cholesterol—differently influence HCN ion channels, which regulate nerve excitability. Changes in these pools after nerve injury alter pacemaker channel behavior, offering insights into pain mechanisms and potential treatments.

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, 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 G-V 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 naïve DRG neurons—characterized by high cholesterol and large OMDs—modulation occurs mostly via increased free cholesterol. These findings provide mechanistic insights into cholesterol-dependent modulation of ion channels and its role in neuropathic pain.