bioRxiv preprint doi: https://doi.org/10.1101/2020.04.30.071076; this version posted May 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kcc2 proteome Isolation and characterization of multi-protein complexes enriched in the K-Cl co-transporter 2 from brain plasma membranes. Joshua L. Smalley1, Georgina Kontou1, Catherine Choi1, Qiu Ren1, David Albrecht1, Krithika Abiraman1, Miguel A. Rodriguez Santos1, Christopher E. Bope1, Tarek Z. Deeb1, Paul A. Davies1, Nicholas J. Brandon2 and Stephen J. Moss*1,3 1Department of Neuroscience, Tufts University School of Medicine, 136 Harrison Ave, Boston 02111. 2Neuroscience, IMED Biotech Unit, AstraZeneca, Boston, MA 02451. 3Department of Neuroscience, Physiology, and Pharmacology, University College, London, WC1E, 6BT, UK. Running Title: Plasma membrane Kcc2 protein complexes and phosphorylation To whom correspondence should be addressed: Professor. S. J. Moss, Department of Neuroscience, Tufts University School of Medicine, Boston MA 02111, Telephone: (617) 636-3976; FAX: (617) 636-2413; Email: [email protected] Key words; Kcc2, proteome, interactome, phosphorylation. ABSTRACT Kcc2 plays a critical role in ankyrins, and the IP3 receptor. We also identified determining the efficacy of synaptic inhibition, interactors more directly associated with however, the cellular mechanism neurons use to phosphorylation; Akap5 and Lmtk3. Finally, we regulate its membrane trafficking, stability and used LC-MS/MS on highly purified endogenous activity are ill-defined. To address these issues, plasma membrane Kcc2 to detect we used affinity purification to isolate stable phosphorylation sites. We detected 11 sites with multi-protein complexes of Kcc2 from the plasma high confidence, including known and novel membrane of murine forebrain. We resolved sites. Collectively our experiments demonstrate these using Blue-native polyacrylamide gel that Kcc2 is associated with components of the electrophoresis (BN-PAGE) coupled to LC- neuronal cytoskeleton and signaling molecules MS/MS. Purified Kcc2 migrated as distinct that may act to regulate transporter membrane molecular species of 300, 600 and 800 kDa trafficking, stability, and activity. following BN-PAGE. In excess of 90% coverage of the soluble N and C-termini of Kcc2 was obtained. The 300kDa species largely contained INTRODUCTION The K+/Cl– co-transporter Kcc2, which is consistent with a dimeric Kcc2 (encoded by the gene SLC12A5) is the quaternary structure for this transporter. principal Cl--extrusion mechanism employed by Intriguingly, lower levels of Kcc1 were also mature neurons in the CNS (1). Its activity is a found in this species suggesting the existence of pre-requisite for the efficacy of fast synaptic “mixed” Kcc2/Kcc1 heterodimers. The 600 and inhibition mediated by Glycine (GLYR) and type A γ-aminobutyric acid receptors (GABA R), 800 kDa species represented stable multi-protein A which are Cl– permeable ligand-gated ion complexes of Kcc2. We identified a set of novel channels. At prenatal and early postnatal stages in structural, ion transporting and signaling protein rodents, neurons have elevated intracellular Cl– interactors, that are present at both excitatory and levels resulting in depolarizing GABAA- inhibitory synapses, consistent with the proposed mediated currents (2). The postnatal development association of Kcc2. These included spectrins, of canonical hyperpolarizing GABAAR currents is a reflection of the progressive decrease of 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.30.071076; this version posted May 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kcc2 proteome intraneuronal Cl– levels that is caused by the for isolating stable multiprotein complexes upregulation of Kcc2 expression and subsequent enriched in this transporter. To do so, fresh activity (3–6). These changes in neuronal Cl– isolated mouse forebrain was homogenized in extrusion reflect a sustained increase in the detergent-free conditions to preserve organelle expression levels of Kcc2 after birth, the mRNA structure. The resulting homogenate was then levels of which do not reach their maximal levels subjected to differential gradient-based in humans until 20-25 years of age (7). In centrifugation and the plasma membrane fraction addition to this, the appropriate developmental was isolated (Figure 1a). To confirm effective appearance of hyperpolarizing GABAAR currents fractionation, we immunoblotted for established is also in part determined by the phosphorylation protein markers for each specific organelle status of Kcc2, a process that facilitates its (Figure 1b); HSP90 (cytosol), HSP60 membrane trafficking and activity (8–13). (mitochondria), calreticulin (ER), and n-cadherin (n-Cadh) (plasma membrane). Each organelle In keeping with its essential role in fraction showed enrichment for its respective determining the efficacy of synaptic inhibition, marker protein. Importantly, the plasma humans with mutations in Kcc2 develop severe membrane fraction was both enriched for n-Cadh epilepsy soon after birth (14–16). Deficits in and depleted for HSP60 compared to the total Kcc2 activity are also believed to contribute to lysate, demonstrating efficient separation of the development of temporal lobe epilepsy (17, plasma membrane and mitochondria, a common 18), in addition to other traumas including contaminant of membranous fractions. Kcc2 was ischemia and neuropathic pain (19, 20). Given the enriched in the ER fraction and highly enriched critical role that Kcc2 plays in determining the in the mitochondrial and plasma membrane maturation of inhibitory neurotransmission, fractions. The enrichment of Kcc2 in our target subtle changes in its function are also strongly fraction - the plasma membrane fraction, was implicated in autism spectrum disorders (ASD) highly encouraging for downstream purification. (21), Down syndrome (22), fragile X syndrome The presence of Kcc2 in the mitochondrial (23), and Rett syndrome (24–26). fraction could be an interesting future area of research, as chloride homeostasis is fundamental Here, we isolated and resolved stable for correct mitochondrial function. protein complexes that contain Kcc2 from highly purified plasma membranes from mouse Following detergent solubilization, forebrain, and characterized their composition membrane fractions were subjected to using an unbiased proteomic approach. We immunoprecipitation with a monoclonal antibody subsequently identified several novel Kcc2- directed against the intracellular C-terminus of associated proteins. We used the same Kcc2 covalently coupled to protein G-coated purification strategy and proteomics to measure ferric beads. Initial optimization experiments Kcc2 phosphorylation. In this way we have revealed that we were able to isolate developed new insights into the potential approximately 65% of the Kcc2 available in the mechanisms of Kcc2 stabilization and regulation lysate and the lysate preparation produced in the plasma membrane along with measuring minimal Kcc2 oligomerization when resolved by the phosphorylation status of Kcc2 on the cell SDS-PAGE (Figure S1a, b). Purified material surface. was eluted under native conditions using a ‘soft elution buffer’ containing 2% Tween-20 (27), and subjected to Blue-Native-PAGE (BN-PAGE; Results Figure 1c). We observed well-resolved bands by colloidal Coomassie staining at 300, 600, and 800 Isolating native stable protein complexes kDa. These bands were not seen in control containing Kcc2 from brain plasma experiments performed with non-immune mouse membranes In order to define which proteins are IgG. An immunoblot of the same sample associated with Kcc2 in the neuronal plasma confirmed that these bands contained Kcc2. membrane we developed a novel methodology Immunoblots of plasma membrane lysates 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.30.071076; this version posted May 1, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Kcc2 proteome separated by BN-PAGE showed that these Analyzing the composition of stable protein distinct Kcc2 complex bands exist in the lysate complexes of Kcc2 Having confirmed the and are not an artifact of or degraded by veracity of our Kcc2 purifications we purification. Despite the high stringency of our investigated the reproducibility of the proteins method (with high speed centrifugation and low associated with Kcc2 in each of the bands. First, concentration SDS exposure), no monomeric we compiled a data matrix of all the proteins and Kcc2 was observed (~150 kDa), indicating that total peptide counts detected by proteomic Kcc2 was maintained in higher order complexes. analysis in each of our samples (Table S1). We This may disrupt low affinity, or transient protein then removed all proteins that were not at least 2- interactions and favor the detection of higher fold enriched in the IP compared to proteins that affinity binding partners. associated with IgG alone (Table S3) and removed all proteins with fewer than 2 peptide After we confirmed that these well counts in each individual proteomic run. This resolved bands contained Kcc2, we assessed their process is illustrated in Figure 2a. Kcc2 content by LC-MS/MS following