BPS2026 – RhoA activation promotes ordered membrane domain coalescence and suppresses neuronal excitability

This study explores how the small GTPase RhoA modulates plasma membrane lipid nanodomains, particularly cholesterol-rich ordered membrane domains (OMDs). These nanodomains play a critical role in regulating ion channel activity and neuronal excitability. However, due to their nanoscale dimensions, OMDs remain challenging to visualize using conventional light microscopy. Here, we used fluorescently labeled cholera toxin B (CTxB) and the lipidated peptides Lck-10 (L10) and Src-15 (S15) as probes to visualize OMDs and quantified their size via Förster resonance energy transfer (FRET)-based confocal fluorescence lifetime imaging microscopy (FLIM). Pharmacological inhibition of RhoA significantly reduced OMD sizes in both human cell lines and nociceptor dorsal root ganglion (DRG) neurons. To achieve better spatiotemporal control of specific RhoA activation, we employed an improved light-inducible dimerization (iLID) system. Optogenetic activation of RhoA rapidly increased FRET efficiency between CTxB probes with a time constant of ∼70 seconds, indicating OMD coalescence. Moreover, disrupting cytoskeletal networks increased the baseline OMD size but abolished RhoA’s effects on OMDs, establishing a link between small GTPase, OMD modulation, and the cytoskeleton. We also found that protein palmitoylation contributed to RhoA’s activation, as its effects were inhibited by 2-bromopalmitate and enhanced by palmitate supplementation. Functionally, RhoA inhibition potentiated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity in nociceptive DRG neurons, increasing action potential firing. Conversely, in a rat model of spared nerve injury (SNI), RhoA activation expanded OMDs in nociceptive DRG neurons and reduced action potential firing in vitro. Constitutive RhoA activation suppressed HCN channel activity in SNI neurons. These findings support a neuroprotective role for RhoA activation, where it restores OMD size and suppresses pathological hyperexcitability in neuropathic pain.