NKCC1-SPAK UNCOUPLING UNCOVERS A NOVEL MECHANISM AND THERAPEUTIC AVENUE FOR KCC2 RESTORATION AND SEIZURE CONTROL

Neuronal chloride homeostasis is governed by the opposing actions of the cation-chloride cotransporters KCC2 and NKCC1, which mediate chloride extrusion and influx and thereby determine the polarity and efficacy of GABAergic neurotransmission. In many neurological disorders, chloride dysregulation is typically associated with NKCC1 upregulation and KCC2 downregulation at the neuronal surface, highlighting KCC2 membrane stability as a critical pathological determinant and therapeutic target. The WNK/SPAK kinase pathway plays a central role in this process by regulating transporter phosphorylation and membrane stability.
Here, we uncover an unexpected mechanism by which NKCC1 governs KCC2 membrane stability. We propose that NKCC1 forms membrane clusters that recruit SPAK and PP1, compensating for the absence of direct SPAK/PP1-binding sites on KCC2. Single-particle tracking reveals that these NKCC1-rich assemblies dynamically trap KCC2, enabling either SPAK-driven phosphorylation and membrane destabilization or PP1-mediated dephosphorylation and stabilization of KCC2.
We further demonstrate that peptides targeting key NKCC1 interaction motifs selectively modulate SPAK activity. Peptides that prevent NKCC1-PP1 interaction reduce KCC2 membrane clustering and chloride extrusion, whereas a peptide that prevents SPAK recruitment to NKCC1 stabilizes KCC2 in membrane clusters and enhances chloride extrusion. Notably, an optimized peptide analog preserves KCC2 clustering under hyperexcitable conditions, reduces seizures in a PTZ-induced epilepsy model, and suppresses ictal activity in human epileptic tissue.
Together, these findings identify NKCC1-KCC2 coupling as a central regulatory axis of inhibitory signaling and establish targeted modulation of NKCC1-SPAK interactions as a promising strategy to restore chloride homeostasis in epilepsy and related disorders.