ANTIBODY-MEDIATED CLUSTERING OF KCC2 AND NKCC1 ENHANCES TRANSPORTER ACTIVITY AND REGULATES NEURONAL CHLORIDE HOMEOSTASIS

Inhibition is essential to prevent pathological states associated with neuronal hyperexcitability and critically depends on intracellular chloride concentration, which determines the efficacy of GABAergic signalling. Chloride homeostasis is regulated by the neuronal cation-chloride cotransporters NKCC1 and KCC2, which mediate chloride influx and extrusion, respectively. In many neurological disorders, increased NKCC1 and decreased KCC2 expression at the neuronal surface identify chloride transporter membrane stability as a key determinant and therapeutic target. NKCC1 and KCC2 are organized into clusters at the neuronal membrane, whose organization is dynamically regulated by a “diffusion–trap” mechanism. However, whether clustering directly enhances membrane stability and transporter function, and whether artificially induced clustering can potentiate chloride transporter activity, remains unclear. Here, we used antibody-mediated cross-linking to generate artificial clusters of KCC2-Flag- and NKCC1-HA-tagged transporters at the surface of cultured hippocampal neurons. Cross-linking in live cells reduced the diffuse transporter pool, indicating recruitment into clusters. It produced larger clusters with higher molecular density, while cluster number decreased, consistent with cluster fusion along dendrites. Using Superclomeleon chloride imaging, we found that KCC2 cross-linking significantly reduced intracellular chloride levels, demonstrating that artificial clustering enhances transporter activity at the neuronal membrane. Together, these findings establish a direct link between chloride transporter clustering and function and identify antibody-mediated clustering as a potential strategy to modulate transporter activity under pathological conditions.