NKCC1 as a signaling hub regulating KCC2 stability, chloride homeostasis, and seizure susceptibility

Chloride homeostasis relies on the dynamic balance between the neuronal co-transporters NKCC1 and KCC2. We reveal an unexpected mechanism by which NKCC1 governs KCC2 membrane stability. NKCC1 clusters recruits SPAK and PP1 to dynamically trap KCC2, compensating for its lack of a direct SPAK-binding site. Single-particle tracking shows that these NKCC1-rich assemblies operate as signaling hubs, enabling either SPAK-driven KCC2 phosphorylation and its membrane destabilization or PP1-mediated dephosphorylation of SPAK and KCC2 membrane stabilization. Peptides that activate SPAK by engaging NKCC1s PP1-binding motif lower KCC2 surface levels and reduce chloride extrusion, whereas a SPAK-inhibiting peptide prevents SPAK recruitment to NKCC1, stabilizes KCC2 in membrane clusters, and enhances chloride extrusion. An optimized peptide analog preserves KCC2 clustering under hyperexcitable conditions, reduces seizure frequency and severity in PTZ-induced epilepsy, and suppresses ictal activity in human epileptic tissue. These findings identify NKCC1-KCC2 coupling as a central regulatory axis for inhibitory signaling, and position our peptides as promising therapeutic candidates to restore chloride homeostasis in epilepsy and other disorders marked by impaired KCC2 membrane stability.