NKCC1 coordinates SPAK–PP1 signalling to regulate KCC2 membrane stability and seizure susceptibility

Chloride homeostasis, and thereby the efficacy of chloride-permeable GABAA receptor signaling, depends on the dynamic balance between NKCC1 (Na+-K+-2Cl- cotransporter 1) and KCC2 (K+-Cl- cotransporter 2), which respectively mediate chloride influx and extrusion to tightly control intracellular chloride concentration ([Cl-]i). Here, we uncover an unexpected mechanism by which NKCC1 regulates KCC2 membrane stability and function. Co-immunoprecipitation experiments from mouse brain lysates revealed that KCC2 associates with NKCC1 and SPAK (STE20/SPS1-related proline-alanine-rich kinase), indicating that these proteins are within a common molecular interaction network. Furthermore, we show that NKCC1 and KCC2 form co-clusters in the plasma membrane of hippocampal neurons and that freely diffusing KCC2 molecules become trapped within NKCC1 membrane clusters. These observations suggest that NKCC1 coordinates SPAK- and PP1-dependent regulation of KCC2. Guided by molecular modeling, we designed peptides targeting specific interactions within this complex. Peptides that activate SPAK by engaging NKCC1's PP1-binding motif decrease KCC2 surface expression and impair chloride extrusion, whereas a SPAK-inhibiting peptide that prevents SPAK recruitment to NKCC1, stabilizes KCC2 in membrane clusters, and enhances chloride extrusion. An optimized peptide analog suitable for in vivo use preserves KCC2 clustering under hyperexcitable conditions, reduces seizure frequency and severity in an acute pentylenetetrazol-induced (PTZ) seizure model, and suppresses ictal activity in chronically epileptic human tissue. Together, these findings identify NKCC1 as a key regulator of KCC2 membrane stability, reveal NKCC1-KCC2 coupling as a critical determinant of inhibitory signaling, and establish this complex as a potential therapeutic target for restoring chloride homeostasis in epilepsy and related neurological disorders.