Abstract
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.
Highlights
GABA and glycine are the key inhibitory neurotransmitters of the mature nervous system, and most synaptic inhibition is mediated by Cl- permeable GABAA and glycine receptors
Upon further analysis using standard SDS-PAGE, we observed that the total KCC2 extracted was greater in C12E9 and CHAPSbased detergent extractions in comparison with all other detergents (Figure 1a, Figure 1—figure supplements 1 and 2), we restricted our further analysis to C12E9 and CHAPS-based membrane preparations
To determine which of these two detergents was optimal for our subsequent full-scale proteomic analysis, we performed affinity purifications (AP)-mass spectrometry (MS) to compare the efficacy of C12E9 versus CHAPS-solubilized membrane fractions
Summary
GABA and glycine are the key inhibitory neurotransmitters of the mature nervous system, and most synaptic inhibition is mediated by Cl- permeable GABAA and glycine receptors. This hyperpolarizing inhibition results from the inward gradient for Cl- established primarily by the K+-Cl- cotransporter KCC2, which exports Cl- to maintain low intracellular Cl- [Cl-]i (Rivera et al, 1999; Doyon et al, 2016). KCC2 is a member of the nine-member family of cation-chloride cotransporters and is unique among the members because it is present exclusively in neurons of the CNS, and mediates the electroneutral outward cotransport of K+ and Cl-.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call