GM1 GANGLIOSIDE CONTROLS KCC2 MEMBRANE ORGANIZATION AND INHIBITORY CHLORIDE TRANSPORT

The neuronal K⁺–Cl⁻ cotransporter KCC2 is essential for inhibitory GABAergic transmission through the maintenance of low intracellular chloride levels. While regulation of KCC2 by protein partners has been extensively studied, the role of membrane lipids in controlling its organization and function remains poorly understood. Here, we identify the ganglioside GM1, a complex lipid especially abundant in plasma membrane of neurons, as a key lipid regulator of KCC2 membrane compartmentalization and stability.
Immunocytochemical and biochemical analyses revealed that KCC2 and GM1 co-localize within neuronal plasma membrane lipid rafts, with their association increasing during postnatal brain maturation. Molecular modeling identified a conserved ganglioside-binding domain (GBD) in KCC2 centered on the tryptophan residue W318. Biophysical assays demonstrated a specific and saturable interaction between GM1 and the KCC2-GBD, which was abolished by the epilepsy-associated W318S mutation. Disruption of this interaction, by the W318S mutation or pharmacological depletion of GM1, prevented KCC2 incorporation into lipid rafts, altered its membrane diffusion, reduced surface clustering, and compromised protein stability.
Functionally, loss of KCC2–GM1 interaction markedly impaired KCC2-mediated Cl⁻ extrusion and collapsed the somato-dendritic chloride gradient in neurons. Importantly, pilocarpine animal models of epilepsy showed a loss of KCC2–GM1 association, indicating disease-related destabilization of this lipid–protein complex. In line with these findings, GM1-deficient mice showed selective downregulation of hippocampal KCC2 expression in vivo.