RhoA regulates membrane order and tension to control excitability of nociceptor neurons

Compartmentalization of the plasma membrane into phase-separated domains has emerged as a fundamental principle in regulating membrane transport and excitability ^1–3 . 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 Förster resonance energy transfer, we quantified nanoscale changes in OMD size in living cells ³ . 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 coalescence ^5–7 . 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.