RhoA activation promotes ordered membrane domain coalescence and suppresses neuronal excitability

Area of Science:

• Cellular Neuroscience
• Membrane Biophysics
• Molecular Signaling

Background:

• Plasma membrane lipid nanodomains, especially cholesterol-rich ordered membrane domains (OMDs), are crucial for regulating ion channel activity and neuronal excitability.
• Visualizing nanoscale OMDs is challenging with conventional microscopy.

Purpose of the Study:

• To investigate the role of the small GTPase RhoA in modulating OMDs.
• To explore the functional consequences of RhoA-mediated OMD changes on neuronal excitability and neuropathic pain.

Main Methods:

• Utilized fluorescently labeled cholera toxin B (CTxB) and Lck-10 (L10) as probes for OMD visualization.
• Quantified OMD size using confocal fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET).
• Employed an improved light-inducible dimerization (iLID) system for optogenetic control of RhoA activation.

Main Results:

• RhoA inhibition reduced OMD size in human cell lines and dorsal root ganglion (DRG) neurons.
• Optogenetic RhoA activation led to rapid OMD coalescence.
• RhoA inhibition potentiated hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity and increased firing in nociceptive DRG neurons.
• In a neuropathic pain model, RhoA activation expanded OMDs, suppressed HCN channel activity, and decreased excitability.

Conclusions:

• RhoA activation modulates OMD size and influences neuronal excitability.
• RhoA activation demonstrates a neuroprotective role by restoring OMD size and reducing pathological hyperexcitability in neuropathic pain.