Direct regulation of the voltage sensor of HCN channels by membrane lipid compartmentalization.

Ion channels function within a membrane environment characterized by dynamic lipid compartmentalization. Limited knowledge exists regarding the response of voltage-gated ion channels to transmembrane potential within distinct membrane compartments. By leveraging fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), we visualized the localization of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in membrane domains. HCN4 exhibits a greater propensity for incorporation into ordered lipid domains compared to HCN1. To investigate the conformational changes of the S4 helix voltage sensor of HCN channels, we used dual stop-codon suppression to incorporate different noncanonical amino acids, orthogonal click chemistry for site-specific fluorescence labeling, and transition metal FLIM-FRET. Remarkably, altered FRET levels were observed between VSD sites within HCN channels upon disruption of membrane domains. We propose that the voltage-sensor rearrangements, directly influenced by membrane lipid domains, can explain the heightened activity of pacemaker HCN channels when localized in cholesterol-poor, disordered lipid domains, leading to membrane hyperexcitability and diseases.

Disruption of Ordered Membrane Domains as a Mechanism Underlying Neuropathic Pain.

Cell membranes consist of heterogeneous lipid domains that influence key cellular processes, including signal transduction, endocytosis, and electrical excitability. The goal of this study was to assess the size of cholesterol-enriched ordered membrane domains (OMD) in various cell types. Multiple cell types were tested using fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET), whereby small nociceptor DRG neurons and cardiac pacemaker cells displayed the highest FRET intensities. This implies that electrically active cells tend to have large OMDs. Treatment of cells with the cholesterol-extracting reagent ß-cyclodextrin (ß-CD) led to a decrease in FRET, indicating a reduction in the OMD size, whereas detergents known to promote domain coalescence in artificial membranes increased OMD size. In an in vitro fatty liver model, palmitate supplementation increased FRET whereas oleate supplementation decreased FRET in isolated primary murine hepatocytes, highlighting the importance of unsaturated lipid tails in lipid domain organization. Disruption of OMD using ß-CD potentiated action potential firing in nociceptor DRG neurons and decreased the free energy needed for opening native hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. After disrupting the OMD, HCN channels exhibited an increased relative open probability at the resting membrane potential (RMP). A significant reduction in FRET was observed in both a chemotherapy-induced neuropathic pain model and a spared nerve injury model of neuropathic pain, consistent with disrupted or shrunken OMD in these models. Collectively, these findings show that disturbances in lipid domains may contribute to the progression of neuropathic pain, and they suggest new therapeutic strategies to achieve pain relief.