RhoA activation promotes ordered membrane domain coalescence and suppresses neuronal excitability

Compartmentalization of the plasma membrane into phase-separated domains has emerged as a fundamental principle in regulating membrane transport and excitability1-3. However, it remains unclear how neuropathic stress initiates lipid remodeling of membrane domains. Here we show a previously unrecognized role for the monomeric GTPase RhoA in driving the coalescence of ordered membrane domains (OMDs)4, coupling cytoskeletal dynamics to membrane fluidity and order. Using FLIM-based Forster resonance energy transfer, we quantified nanoscale changes in OMD size in living cells3. Pharmacological modulation of RhoA activity altered OMD size in both human cell lines and nociceptor dorsal root ganglion (DRG) neurons. Optogenetic activation of RhoA using an improved light-inducible dimerization system triggered rapid OMD coalescence5-7. Moreover, RhoA-mediated remodeling of OMDs required an intact cytoskeletal network and was driven by heightened membrane lateral tension, a response that was also dependent on protein palmitoylation. Functionally, RhoA inhibition increased action potential firing and potentiated pacemaker HCN channel activity in nociceptive DRG neurons. Conversely, in a spared nerve injury model--where DRG neurons display small OMDs, reduced membrane tension, and hyperexcitability--RhoA activation enlarged OMDs, suppressed HCN channel activity, and dampened neuronal excitability. Together, these findings suggest RhoA-driven OMD remodeling as a key adaptive mechanism that counteracts the hyperexcitability associated with neuropathic pain. They further highlight reduced membrane tension as a biophysical signature of neuropathic stress and suggest that targeting the RhoA pathway may offer a therapeutic strategy for chronic pain.