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

  1. Vivek Mahadevan
  2. C Sahara Khademullah
  3. Zahra Dargaei
  4. Jonah Chevrier
  5. Pavel Uvarov
  6. Julian Kwan
  7. Richard D Bagshaw
  8. Tony Pawson
  9. Andrew Emili
  10. Yves De Koninck
  11. Victor Anggono
  12. Matti Airaksinen
  13. Melanie A Woodin  Is a corresponding author
  1. University of Toronto, Canada
  2. University of Helsinki, Finland
  3. Mount Sinai Hospital, Canada
  4. Institut Universitaire en Santé Mentale de Québec, Canada
  5. Université Laval, Canada
  6. The University of Queensland, Australia
Research Article
Cite this article as: eLife 2017;6:e28270 doi: 10.7554/eLife.28270
6 figures, 1 table, 1 data set and 1 additional file

Figures

Figure 1 with 2 supplements
KCC2 multi-protein complexes can be extracted using native detergents.

(a) BN-PAGE and SDS-PAGE separation of solubilized membrane fractions prepared from ~P50 mouse brain, using the detergents listed in the associated table. Protein separations were western-blotted and probed with antibodies indicated on the left. O, oligomer; M, monomer. Blots are representative of two independent biological replicates. (b) Comparison of the top 35 proteins identified with high confidence in C-terminal KCC2 antibody immunoprecipitations from CHAPS-based or C12E9-based membrane extractions. IgG-AP immunoprecipitations were performed as a control. Heat maps represent log scale spectral counts of individual proteins per condition, expressed relative to global spectral counts. Unique peptides corresponding to KCC2 (indicated in red font) were most abundant in both conditions, confirming the specificity of the C-terminal antibody. Previously identified KCC2 interacting partners are identified by asterisks. Proteins in green represent those that commonly co-precipitated with KCC2 regardless of the detergent extraction.

https://doi.org/10.7554/eLife.28270.003
Figure 1—source data 1

Proteins enriched in KCC2-AP using CHAPS vs C12E9.

https://doi.org/10.7554/eLife.28270.006
Figure 1—figure supplement 1
Workflow to enrich KCC2 complexes.

SDS, sodium dodecyl sulfate; DOC, deoxycholate; NP40, Igepal-CA630; C12E9, nonaethylene glycol monododecyl ether; DDM, n-dodecyl-β-D-maltoside; PFO, perfluoro-octanoic acid; IAA, Iodoacetmide; BN, blue-native; SB, sample buffer for gel loading. RED/ORANGE lines and boxes indicate harsh KCC2 extraction conditions; YELLOW lines and boxes indicate intermediary KCC2 extraction conditions; BLUE lines and boxes indicate mild, native-KCC2 extraction conditions. The orange and yellow extraction/gel running strategies were employed for studying the stability of KCC2 oligomers (by subjecting them to harsh-to-mildly denaturing conditions). The blue extraction/gel running conditions were employed to study the composition of native-KCC2-oligomers.

https://doi.org/10.7554/eLife.28270.004
Figure 1—figure supplement 2
SDS-PAGE separation of solubilized membrane fractions.

Obtained with high-salt Tris-HCl buffer containing the following detergents (lane1: 1%Triton, 1%DOC; lane 2: 0.1%SDS, 0.5%DOC, 1%NP40 (RIPA); lane 3: 1%NP40 and lane 4: 1.5% C12E9). Samples were denatured in SDS-sample buffer containing 100 mM DTT or 25 mM DTT, @ 37C for 30 min. Yellow-boxes indicate that C12E9-based native detergent enriches for more putative-KCC2 oligomers than the previously published KCC2 detergent extractions (lanes 1–3); and that the putative KCC2 oligomers are DTT-sensitive.

https://doi.org/10.7554/eLife.28270.005
Figure 2 with 1 supplement
Multi-epitope AP identifies native-KCC2 protein constituents in mouse brain.

(a) Schematic of the locations of anti-KCC2 antibodies. (b) The primary KCC2 amino acid sequence. Red indicates the protein coverage of KCC2 identified by MS analysis; yellow indicates unique coverage for KCC2a and KCC2b isoforms. MS/MS- spectra of peptides unique for KCC2a and KCC2b. Right: the MS/MS ion fragmentation of the corresponding amino acid sequence is indicated above the spectra. (c) Spectral and peptide count plots of proteins in AP with all three anti-KCC2 antibodies in developing brain membrane fractions (P5, left) and adult brain membrane fractions (P50, right). Peptide and spectral counts are normalized (anti-KCC2/IgG) and plotted on a log scale. Red circles - highly enriched KCC2 bait. Blue circles - highly enriched PACSIN1 target peptides. Dark-grey circles - top proteins enriched with KCC2-AP in comparison to IgG control-AP. Light-grey circles - proteins enriched in IgG control-AP in comparison to KCC2-AP and known spurious interactors.

https://doi.org/10.7554/eLife.28270.007
Figure 2—figure supplement 1
Validation of KCC2 antibodies for immunodepletion.

Native KCC2 complexes from C12E9-solubilized whole-brain membrane fractions immunoprecipitated with pre-immune sera or anti-N-terminal KCC2b antibody (a) or anti-pSer940 KCC2 antibody (b) and immunoblotted with the antibodies indicated at right (C-terminal KCC2 antibody). Representative example of five biological replicates. IP, immunoprecipitate; I, input fraction (1% of IP); U. unbound fraction (1%of IP); O. oligomer; M. monomer.

https://doi.org/10.7554/eLife.28270.008
Figure 3 with 2 supplements
ME-AP reveals distinct KCC2 constituents in developing and mature brain.

Summary of the top 70 proteins identified with high confidence across KCC2-ME AP in the developing and mature brain. PLATINUM interactors: proteins enriched in a minimum of 2/3 replicates, and show 5 + fold spectral enrichment. GOLD interactors: proteins with 5 + fold spectral enrichment in one replicate. SILVER interactors: 3–5 fold spectral enrichment; BRONZE interactors: 1.5–3 fold spectral enrichment. Enrichment is in KCC2-AP in comparison with IgG-AP. Heat map represents log scale spectral enrichment of individual proteins per antibody condition, relative to respective control conditions. See Table 1 for a list of the transmembrane and soluble KCC2 interactors. .

https://doi.org/10.7554/eLife.28270.010
Figure 3—source data 1

All validated peptide-spectrum matches for KCC2-AP and IgG-AP.

https://doi.org/10.7554/eLife.28270.013
Figure 3—source data 2

Top CRAPome members that appear at 50% frequency.

https://doi.org/10.7554/eLife.28270.014
Figure 3—source data 3

KCC2 interactors identified in AP/MS categorized into platinum, gold, silver, and bronze.

https://doi.org/10.7554/eLife.28270.015
Figure 3—source data 4

Previously established KCC2 partners not identified in the present screen.

https://doi.org/10.7554/eLife.28270.016
Figure 3—figure supplement 1
ME-AP proteomics identify the protein constituents of native KCC2.

(a) Venn diagram comparison of the intersection of data obtained using N-term and C-term antibodies in developing and mature brain. (b) Proteins that appear exclusively with P5 or P50 KCC2-immunoprecipitates. (c) Similar to (a) but for data obtained using all three antibodies. (d) Proteins that appear exclusively to the individual KCC2 antibody.

https://doi.org/10.7554/eLife.28270.011
Figure 3—figure supplement 2
Workflow for curating the KCC2 interactome.
https://doi.org/10.7554/eLife.28270.012
Figure 4 with 1 supplement
Members of the KCC2 interactome are highly represented at excitatory synapses.

The KCC2 interactome mapped to (a) the excitatory synapse-enriched proteomes or, (b) the inhibitory synapse-enriched proteomes. The thickness of the black radial lines in the foreground denotes the number of spectral enrichment (KCC2/IgG) in the log scale. Grey radial lines in the background denotes the previously identified physical/co-expression networks across all interactome members. IPA revealing the members of the KCC2 interactome that are involved in (c) ion homeostasis, (d) dendritic cytoskeleton rearrangement and (e) recycling/endocytosis/trafficking. The following source data and figure supplements are available for Figure 4.

https://doi.org/10.7554/eLife.28270.018
Figure 4—source data 1

Interactome mapping at excitatory and inhibitory synapses.

https://doi.org/10.7554/eLife.28270.020
Figure 4—source data 2

Ingenuity pathway analysis.

https://doi.org/10.7554/eLife.28270.021
Figure 4—figure supplement 1
The SLC12A5/KCC2 interactome.

The KCC2 interactome was mapped to the excitatory synapse-enriched proteomes, and the inhibitory synapse-enriched proteomes. Circle/triangle/square-shaped nodes represent the KCC2 partners identified in this present study; diamond-shaped nodes represent the KCC2 partners not identified, but previously established as physical/functional partners of KCC2. Red/blue/pink-filled nodes represent synaptic-KCC2 partners; uncolored nodes represent the putative-, non-synaptic KCC2 partners. The thickness of the radial lines represents the spectral enrichment (KCC2/IgG). See Figure 4—source data 1 for the complete list of all proteins used for mapping.

https://doi.org/10.7554/eLife.28270.019
Figure 5 with 2 supplements
Characterization of the PACSIN1-KCC2 interaction.

(a) Spatiotemporal expression patterns of SLC12A5 and members of receptor trafficking node of the KCC2 interactome in the human brain; RNAseq data were analyzed in hippocampus. Pcw, postconceptual weeks. (b) Native KCC2 complexes from C12E9-solubilized whole-brain membrane fractions immunoprecipitated with IgY or anti-N-term KCC2 (left) and IgG or anti-PACSIN1 (right), and immunoblotted with antibodies as indicated. (c) Western blot analysis of developmental expression patterns of KCC2 and PACSIN1. (d) Antibody-shift assay followed by 2D-BN-PAGE separation using whole-brain membrane fractions, incubated with antibodies indicated on left; gel separations were immunoblotted with anti-KCC2 or PACSIN1 antibodies. (e) Coimmunoprecipitation performed in COS7 cells transfected with myc-tagged KCC2b and GFP-tagged PACSIN1/2/3 constructs, immunoprecipitated with anti-N-term KCC2b, and immunoblotted with the antibodies indicated at right. (f) Immunoblot of immunoprecipitates from transfected COS7 cell lysates. * indicate the lanes where PACSIN1 lacks the variable region between ~aa325-383. # of independent biological replicates are indicated in parenthesis: Figure 5e (4), Figure 5f (3), Figures 5b,c,d (2).

https://doi.org/10.7554/eLife.28270.022
Figure 5—source data 1

Human brain RNAseq data from the Allan Brain Atlas for receptor trafficking node.

https://doi.org/10.7554/eLife.28270.025
Figure 5—figure supplement 1
Spatiotemporal expression patterns of SLC12A5 and members of receptor trafficking node of the KCC2 interactome in the human brain.

The RNAseq data were analyzed for the above members across five brain regions including Amygdala, Striatum, Thalamus, Cerebellum, and the ganglionic eminences at eight different developmental periods.

https://doi.org/10.7554/eLife.28270.023
Figure 5—figure supplement 2
The primary amino acid sequence coverage of PACSIN1 (left), and protein coverage of PACSIN1 identified by MS analysis are indicated in red.

MS/MS- spectra of a peptide unique for PACSIN1, highlighted in yellow. The MS/MS ion fragmentation of the corresponding amino acid sequence is indicated above the spectra (right).

https://doi.org/10.7554/eLife.28270.024
Figure 6 with 1 supplement
PACSIN1 is a negative regulator of KCC2 expression and function.

(a) Example IV curves measuring EGABA using Cl--loading through whole-cell configuration from cultured hippocampal neurons. Neurons were transduced with either control shRNA (n = 9; left) or PACSIN1 shRNA (n = 11; right). Summary of (b) EGABA, (c) synaptic conductance, and (d) RMP from all experiments similar to the examples in a. (e) Summary of EGABA recordings obtained by gramicidin-perforated patch clamp recordings. (f) Example confocal microscopic immunofluorescent images from cultured hippocampal neurons transduced with control shRNA (n = 32) or PACSIN1 shRNA (n = 32) and stained with anti-KCC2 (red; scale bar, 10 μm); green immunostain reports transfection. Below: summary of fluorescence intensities. (g) Similar to f, except neurons were transduced with either control eGFP (n = 23) or PACSIN1-eGFP (n = 16). n values for all experiments on cultured neurons were obtained from a minimum of three independent sets of cultures. Statistical significance was determined using student’s t-tests (two-tailed); *p<0.05, ***p<0.001. For all summary plots, the error bars denote mean ± sem. The following figure supplements is available for Figure 6.

https://doi.org/10.7554/eLife.28270.026
Figure 6—figure supplement 1
Example illustrating the calculation of fluorescence intensity.

Left, using ImageJ, four bisecting lines were drawn across the center of the cell. Right, the peak values of each line (2 values/line) were used to calculate peak fluorescent intensity of KCC2 at the membrane.

https://doi.org/10.7554/eLife.28270.027

Tables

Table 1
KCC2 protein partners identified by ME-APs
https://doi.org/10.7554/eLife.28270.017
Protein nameUniProt IDSpectral ratioMaxPESISP5P50pS940
KCC2bQ91V14-2349.01XXXXX
KCC2aQ91V14-1203.81XXXXX
PACSIN1Q61644136.01XXXXX
SLC44A1Q6X89354.01X
ATP2B4Q6Q47744.01XX
KIF21BQ9QXL141.01X
VCPQ0185324.01XX
GLSD3Z7P323.51XX
RAB11FIP5Q8R36122.01XXX
SRCIN1Q9QWI617.01XXX
RNF8Q8VC5616.01X
JAGN1Q5XKN412.01X
PPFIA3P6046911.01XXX
SLC2A1P1780911.00.96X
YWHAEP6225910.00.99XXXXX
ACTN4P577809.01XX
CRMP1P974278.01XXXX
PITPNM2Q6ZPQ68.01XX
ACO2Q99KI07.01XXXX
COX4I1P197836.00.96X
SLC4A10Q5DTL96.01XXX
USP24B1AY136.01X
YWHAGP619826.00.96XXXX
C1QBP141065.00.89XX
CCT6AP803175.00.99XX
CPSF6Q6NVF95.00.99X
DDX5Q616565.00.99XX
DYNC1LI1Q8R1Q85.00.78XXX
SEC23AQ014055.00.99XXX
SLC8A2Q8K5965.00.99XX
TRIM3Q9R1R25.00.78XXXX
CANXP355644.51XXXX
ATP5HQ9DCX24.00.81X
ATP6V1E1P505184.00.78XXX
DDX17Q501J64.00.96X
DNAJA3Q99M874.00.78XXX
DPYSL3Q621884.00.96X
EWSR1Q615454.01XXX
MAGP209174.00.96XXX
MDH2P082494.00.96XX
RTCBQ99LF44.00.98XX
SNRPAQ621894.00.96X
STOML2Q99JB24.00.96XX
STRN3Q9ERG24.00.96X
YWHAQP682544.00.78XX
INAP466603.61XX
ATP6V1FQ9D1K23.50.95XX
DCTN2Q99KJ83.51XXX
CD47Q617353.20.9XX
ACOX3Q9EPL93.00.78X
AP2B1Q9DBG33.01XXX
APBA1B2RUJ53.00.78X
ATP2A2O551433.00.81XXX
CMPK1Q9DBP53.00.78XX
CRTC1Q68ED73.00.78X
CTNNA2Q613013.00.78XX
FLIIQ9JJ283.00.78XX
FXYD7P596483.00.78X
GPM6AP358023.00.93XXX
GRID2Q616253.00.78XX
NARSQ8BP473.00.78X
NDUFA2Q9CQ753.01X
NUDT21Q9CQF33.00.78X
PPP1CAP621373.00.78XXX
PPP2CAP633303.00.78XX
PRRT2E9PUL53.00.78XXX
SFPQQ8VIJ63.01XXX
SIRT2Q8VDQ83.00.89XX
SLC1A2P430063.00.69XXX
TCP1P119833.00.78XX
TRIOQ0KL023.00.78XX
PTNP630892.71X
RAB2AP539942.70.84XX
NDUFS5Q99LY92.50.82X
CCT2P803142.51XX
DNM1P390532.51XX
SLC25A11Q9CR622.50.93XXX
DDX1Q91VR52.40.89XXX
NEDD4LQ8CFI02.40.91XXXX
SYNGR3Q8R1912.41XXX
DLD*O087492.30.44XXXX
SNAP25P608792.30.65XXXX
DDX3XQ621672.30.79XXXX
CAMK2GQ923T92.31XX
FASNP190962.31XXX
PKMP524802.20.85X
NDUFA9Q9DC692.10.96XX
BASP1Q91XV32.10.72XXX
CKBQ044472.00.64XX
COX6CQ9CPQ12.00.89X
CSNK2A1Q607372.00.84XXX
DHX9*O701332.00.44XX
DPYSL2O085532.00.97XXXX
EDC4Q3UJB92.01X
FUSP569592.00.94XXXX
KCNAB2P624822.00.92XX
NDUFA8Q9DCJ52.00.96XX
NDUFS8Q8K3J12.01X
PDIA6Q922R82.00.89XX
SFXN3*Q91V612.00.44XX
SLC25A22Q9D6M32.00.89XX
STMN2P558212.01X
TNRQ8BYI92.01XXX
TUBB4BP683721.90.55XX
ATP5C1Q91VR21.90.6XX
PPIAP177421.80.74XX
CKMT1P302751.80.86XXXX
COX5AP127871.80.54X
C1QC*Q021051.80.43XX
NDUFS1Q91VD91.80.88XXX
WWP1Q8BZZ31.80.88X
ATP5BP564801.70.38XXXX
CCT5P803161.70.81XXX
DCLK1Q9JLM81.70.96XXX
SLC25A3Q8VEM81.71XXX
SPTBN1Q622611.70.7XX
TUBB3Q9ERD71.60.73XXX
CAMK2DQ6PHZ21.60.82XXX
ATP6V0A1Q9Z1G41.50.92XX
CFL1*P187601.50.44XXX
ATP1A2*Q6PIE51.50.04XXXX
ADGRL2Q8JZZ71.50.89X
BSNO887371.50.72XX
DBTP533951.50.86XXX
GTF2IQ9ESZ81.50.89X
HELBQ6NVF41.50.89X
HK1P177101.50.89XX
HMCN2A2AJ761.50.89X
LGI3Q8K4061.50.89X
PCQ059201.50.89X
RAB3IPQ68EF01.50.89X
UQCR11Q9CPX81.50.89X
ATP1A1Q8VDN21.50.67XXXX
ATP5OQ9DB201.50.71XX
NDUFA4Q624251.40.51XXX
ATP6V0D1P518631.40.53XX
ACAT1*Q8QZT11.40.43XXX
ATP2B2*Q9R0K71.40.47XX
TUBB4AQ9D6F91.40.64XXX
GNB1P628741.40.58XXXXX
C1QAP980861.40.36XX
NDUFA10Q99LC31.40.65XX
RAP2B*P612261.30.42XX
SYT1P460961.30.65XXX
SLC1A3P565641.30.9XXX
CAPRIN1Q608651.30.56XX
YWHAZ*P631011.30.44XXXXX
ATP1A3*Q6PIC61.30XXXX
STX1B*P612641.30.45XXXX
NEFMP085531.20.78XX
DLSTQ9D2G21.20.72XX
  1. Orange-Transmembrane.

    Grey-Soluble.

  2. Green-Secreted/Extracellular.

    ES – excitatory synapse.

  3. IS – inhibitory synapse.

Data availability

The following data sets were generated
  1. 1
    KCC2 Interactome
    1. Mahadevan V
    2. Woodin MA
    (2017)
    Publicly available at EBI PRIDE Archive (accession no: PXD006046).

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