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KCC2 is a neuron-specific K+-Cl– cotransporter essential for establishing the Cl- gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using 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 diverse 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 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., eLife digest Neurons in the brain talk to each other by releasing chemicals called neurotransmitters. These neurotransmitters can either increase ('excite') or decrease ('inhibit') the activity of other neurons. Inhibitory neurotransmission uses the chemical GABA as a neurotransmitter. When a neuron releases GABA it is like applying the brake in your car – you can slow down subtly to stay under the speed limit, or stomp on it to avoid an accident. The brain needs to carefully control the amount of inhibition so that the animal can learn and produce complex behaviours. For GABA to inhibit the activity of a neuron, the neuron must maintain a low amount of chloride ions inside. A transporter protein called KCC2 shuttles chloride out of cells; if this transporter fails to work, chloride builds up in the neuron and prevents inhibition so that GABA neurotransmission switches from inhibitory to excitatory. This breakdown of GABA inhibition is a hallmark of abnormal brain activity during conditions such as epilepsy, pain and some forms of autism. Despite the fact that neurons need KCC2 for inhibition in the brain, we do not know much about how this transporter works. Since the activity of a protein is determined in part by the other proteins it interacts with, it is therefore important to identify all the proteins that interact with KCC2 – termed the KCC2 interactome. To discover these protein interactions, Mahadevan et al. performed a technique called liquid chromatography-mass spectrometry on KCC2 protein isolated from mouse brains. This revealed that there are 181 proteins in the KCC2 interactome. Of these proteins, the most abundant was a protein called PACSIN1, which helps to pull proteins out of the membrane that surrounds each neuron. To investigate how the interaction between PACSIN1 and KCC2 regulates the activity of this transporter, Mahadevan et al. performed fluorescence imaging of neurons and recorded their electrical activity. This revealed that PACSIN1 restricts the expression of KCC2, meaning that the more PACSIN there is in the neuron, the less KCC2 will be present. The KCC2 interactome provides a database of proteins that can be targeted to increase the activity of KCC2. This could allow new treatments to be developed for brain disorders in which the inhibition of neurons is reduced.