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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 na\u00efve DRG neurons\u2014characterized by high cholesterol and large OMDs\u2014modulation occurs mostly via increased free cholesterol. These findings provide mechanistic insights into cholesterol-dependent modulation of ion channels and its role in neuropathic pain.<\/jats:p>","DOI":"10.1085\/jgp.202513925","type":"journal-article","created":{"date-parts":[[2026,2,20]],"date-time":"2026-02-20T07:20:34Z","timestamp":1771572034000},"update-policy":"https:\/\/doi.org\/10.1085\/jgp.crossmarkpolicy","source":"Crossref","is-referenced-by-count":3,"title":["Sequential membrane remodeling by cholesterol distinctly modulates HCN channels in na\u00efve and neuropathic DRG neurons"],"prefix":"10.1085","volume":"158","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-0960-742X","authenticated-orcid":true,"given":"Lucas J.","family":"Handlin","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/01p7jjy08","id-type":"ROR","asserted-by":"publisher"}],"name":"Saint Louis University School of Medicine 1 Edward A. 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