Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

Abstract

KCC2 is a neuron-specific K⁺-Cl⁻ cotransporter essential for establishing the Cl⁻ gradient required for hyperpolarizing 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 and neuropathic pain. Using unbiased functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with a diverse set of proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. 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.