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Native KCC2 Interactome Reveals PACSIN1 As a Critical Regulator of Synaptic Inhibition

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Native KCC2 Interactome Reveals PACSIN1 As a Critical Regulator of Synaptic Inhibition bioRxiv preprint doi: https://doi.org/10.1101/142265; this version posted May 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition Vivek Mahadevan1, 2, C. Sahara Khademullah1, #, Zahra Dargaei1, #, Jonah Chevrier1,, Pavel Uvarov3, Julian Kwan4, Richard D. Bagshaw5, Tony Pawson5, §, Andrew Emili4, Yves DeKoninck6, Victor Anggono7, Matti S. Airaksinen3, Melanie A. Woodin1* 1 Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, Ontario, M5S 3G5, Canada 2 Present address: Section on Cellular and Synaptic Physiology, NICHD, Bethesda, Maryland, USA 3 Department of Anatomy, Faculty of Medicine, University of Helsinki, Finland 4 Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, M5S 3E1, Canada 5 Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada 6 Institut Universitaire en Santé Mentale de Québec, Québec, QC G1J 2G3, Canada; Department of Psychiatry and Neuroscience, Université Laval, Québec, QC, G1V 0A6, Canada 7 Queensland Brain Institute, Clem Jones Centre for Ageing Dementia Research, The University of Queensland, Brisbane, QLD 4072, Australia # Equal contribution § Deceased August 7, 2013 * Corresponding author: [email protected], 416-978-8646 bioRxiv preprint doi: https://doi.org/10.1101/142265; this version posted May 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Abstract 2 KCC2 is a neuron-specific K+-Cl‑ cotransporter essential for establishing the Cl- gradient required for 3 hyperpolarizing inhibition. KCC2 is highly localized to excitatory synapses where it regulates spine 4 morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to numerous human 5 neurological disorders including epilepsy and neuropathic pain. Using unbiased functional proteomics, we 6 identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate 7 KCC2 function. Our analysis revealed that KCC2 interacts with a diverse set of proteins, and its most 8 predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 9 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the 10 PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in 11 hippocampal neurons significantly increases KCC2 expression and hyperpolarizes the reversal potential 12 for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 13 as a novel and potent negative regulator of KCC2. 14 15 Introduction 16 GABA and glycine are the key inhibitory neurotransmitters of the mature nervous system, and most synaptic - 17 inhibition is mediated by Cl permeable GABAA and glycine receptors. This hyperpolarizing inhibition results 18 from the inward gradient for Cl- established primarily by the K+-Cl- cotransporter KCC2, which exports Cl- 19 to maintain low intracellular Cl- (Rivera et al., 1999; Doyon et al., 2016). KCC2 is a member of the SLC12A 20 family of cation-chloride cotransporters and is unique among the members because it is present exclusively 21 in neurons, and mediates the electroneutral outward cotransport of K+ and Cl-. 22 During embryonic development KCC2 expression is low and GABA and glycine act as excitatory 23 neurotransmitters, however during early postnatal development KCC2 expression is dramatically 24 upregulated and GABA and glycine become inhibitory (Ben-Ari, 2002; Blaesse et al., 2009). Excitation- 25 inhibition imbalance underlies numerous neurological disorders (Kahle et al., 2008; Nelson and Valakh, 26 2015), and in many of these disorders, the decrease in inhibition results from a reduction in KCC2 27 expression. In particular, KCC2 dysfunction contributes to the onset of seizures (Huberfeld et al., 2007; 28 Kahle et al., 2014; Puskarjov et al., 2014; Stödberg et al., 2015; Saitsu et al., 2016), neuropathic pain (Coull 2 bioRxiv preprint doi: https://doi.org/10.1101/142265; this version posted May 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Mahadevan et al 2017 Native KCC2 Interactome 29 et al., 2003), schizophrenia (Tao et al., 2012), and autism spectrum disorders (ASD) (Cellot and Cherubini, 30 2014; Tang et al., 2015; Banerjee et al., 2016). Despite the critical importance of this transporter in 31 maintaining inhibition and proper brain function, our understanding of KCC2 regulation is rudimentary. 32 In addition to its canonical role of Cl- extrusion that regulates synaptic inhibition, KCC2 has also emerged 33 as a key regulator of excitatory synaptic transmission. KCC2 is highly localized in the vicinity of excitatory 34 synapses (Gulyas et al., 2001; Chamma et al., 2013) and regulates both the development of dendritic spine 35 morphology (Li et al., 2007; Chevy et al., 2015; Llano et al., 2015) and function of AMPA-mediated 36 glutamatergic synapses (Gauvain et al., 2011; Chevy et al., 2015; Llano et al., 2015). Thus, a dysregulation 37 of these non-canonical KCC2 functions at excitatory synapses may also contribute to the onset of 38 neurological disorders associated with both KCC2 dysfunction and excitation-inhibition imbalances. 39 KCC2 is regulated by multiple posttranslational mechanisms including phosphoregulation by distinct 40 kinases and phosphatases (Lee et al., 2007; Kahle et al., 2013; Medina et al., 2014), lipid rafts and 41 oligomerization (Blaesse et al., 2006; Watanabe et al., 2009), and protease-dependent cleavage (Puskarjov 42 et al., 2012). KCC2 expression and function is also regulated by protein interactions, including creatine 43 kinase B (CKB) (Inoue et al., 2006), sodium/potassium ATPase subunit 2 (ATP1A2) (Ikeda et al., 2004), 44 chloride cotransporter interacting protein 1 (CIP1) (Wenz et al., 2009), protein associated with Myc (PAM) 45 (Garbarini and Delpire, 2008), 4.1N (Li et al., 2007), the glutamate receptor subunit GluK2, its auxiliary 46 subunit Neto2 (Ivakine et al., 2013; Mahadevan et al., 2014), cofilin1 (CFL1) (Chevy et al., 2015; Llano et 47 al., 2015) and RAB11(Roussa et al., 2016). However, since KCC2 exists in a large multi-protein complex 48 (MPC) (Mahadevan et al., 2015), it is likely that these previously identified interactions do not represent all 49 of the components of native-KCC2 MPCs. 50 In the present study, we performed unbiased multi-epitope tagged affinity purifications (ME-AP) of 51 native-KCC2 coupled with high-resolution mass spectrometry (MS) from whole-brain membrane fractions 52 prepared from developing and mature mouse brain. We found that native KCC2 exists in macromolecular 53 complexes comprised of interacting partners from diverse classes of transmembrane and soluble proteins. 54 Subsequent network analysis revealed numerous previously unknown native-KCC2 protein interactors 55 related to receptor recycling and vesicular endocytosis functions. We characterized the highest-confidence 3 bioRxiv preprint doi: https://doi.org/10.1101/142265; this version posted May 25, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. Mahadevan et al 2017 Native KCC2 Interactome 56 KCC2 partner identified in this screen, PACSIN1, and determined that PACSIN1 is a novel and potent 57 negative regulator of KCC2 function. 58 59 RESULTS 60 Determining Affinity Purification (AP) conditions to extract native-KCC2 61 In order to determine the composition of native KCC2 MPCs using AP-MS, we first determined the 62 detergent-based conditions that preserve native KCC2 following membrane extraction. In a non-denaturing 63 Blue-Native PAGE (BN-PAGE), native-KCC2 migrated between 400 kDa – 1000 kDa in the presence of 64 the native detergents C12E9, CHAPS, and DDM. However, all other detergent compositions previously used 65 for KCC2 solubilization resulted in KCC2 migration at lower molecular weights (Figure 1a). This indicates 66 that native detergent extractions are efficient at preserving higher-order KCC2 MPCs. Upon further analysis 67 using standard SDS-PAGE we observed that the total KCC2 extracted was greater in C12E9 and CHAPS- 68 based detergent extractions in comparison with all other detergents (Figure 1a, Figure 1 – Figure 69 Supplement 1, 2), hence we restricted our further analysis to C12E9 and CHAPS-based membrane 70 preparations. 71 To determine which of these two detergents was optimal for our subsequent full-scale proteomic 72 analysis, we performed AP-MS to compare the efficacy of C12E9 versus CHAPS-solubilized membrane 73 fractions. Immunopurification was performed on membrane fractions prepared from adult (P50) wild-type 74 (WT) mouse brain, using a well-validated commercially
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