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Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

GABAA receptor-mediated currents shift from excitatory to inhibitory during postnatal brain development in rodents. A postnatal increase in KCC2 protein expression is considered to be the sole mechanism controlling the developmental onset of hyperpolarizing synaptic transmission, but here we identify a key role for KCC2 phosphorylation in the developmental EGABA shift. Preventing phosphorylation of KCC2 in vivo at either residue serine 940 (S940), or at residues threonine 906 and threonine 1007 (T906/T1007), delayed or accelerated the postnatal onset of KCC2 function, respectively. Several models of neurodevelopmental disorders including Rett syndrome, Fragile × and Down’s syndrome exhibit delayed postnatal onset of hyperpolarizing GABAergic inhibition, but whether the timing of the onset of hyperpolarizing synaptic inhibition during development plays a role in establishing adulthood cognitive function is unknown; we have used the distinct KCC2-S940A and KCC2-T906A/T1007A knock-in mouse models to address this issue. Altering KCC2 function resulted in long-term abnormalities in social behavior and memory retention. Tight regulation of KCC2 phosphorylation is therefore required for the typical timing of the developmental onset of hyperpolarizing synaptic inhibition, and it plays a fundamental role in the regulation of adulthood cognitive function.