KCC2 is a neuron-specific K+-Cl- cotransporter essential for establishing the Cl- gradient required for fast hyperpolarizing GABAergic and glycinergic inhibition. KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to numerous human neurological disorders including epilepsy, neuropathic pain, schizophrenia and autism spectrum disorders. In the present study, we performed unbiased affinity purifications of native-KCC2 coupled with high-resolution mass spectrometry using three different KCC2 epitopes from whole-brain membrane fractions prepared from developing (P5) and mature mouse brain (P50).
We determined that the mouse brain KCC2 functional interactome is comprised of 181 proteins belonging to diverse classes of transmembrane and soluble proteins; and by mapping the KCC2 interactome to excitatory and inhibitory synapse proteomes and performing ingenuity pathway analysis, we determined that KCC2 partners are highly enriched at excitatory synapses and form a dense network with proteins involved in receptor trafficking. Notably, this analysis revealed multiple novel putative-KCC2 partners, including PACSIN1, SNAP25, RAB11FIP5, CSNK2A1, DNM1 and AP2B1. All of these novel putative interactors have established functions in membrane recycling and/or trafficking of distinct glutamate receptor subunits.
The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons significantly increases KCC2 expression and hyperpolarizes the reversal potential for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.
In conclusion, the KCC2 interactome as presented here, serves as a molecular framework for systematically exploring how KCC2 up and down states can be dynamically regulated by its native molecular constituents, thereby providing a blueprint for subsequent detailed functional investigations.