Figures and data in Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition

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6 figures, 1 table and 1 additional file

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Figure 1 with 2 supplements

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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
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Figure 1—figure supplement 1

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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

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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, @ 37^⁰C 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

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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—source data 1 LC/MS replicates.: https://doi.org/10.7554/eLife.28270.009
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Figure 2—figure supplement 1

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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-p^Ser940 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

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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
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Figure 3—source data 2 Top CRAPome members that appear at 50% frequency.: https://doi.org/10.7554/eLife.28270.014
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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
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Figure 3—source data 4 Previously established KCC2 partners not identified in the present screen.: https://doi.org/10.7554/eLife.28270.016
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Figure 3—figure supplement 1

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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

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Workflow for curating the KCC2 interactome.
https://doi.org/10.7554/eLife.28270.012

Figure 4 with 1 supplement

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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
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Figure 4—source data 2 Ingenuity pathway analysis.: https://doi.org/10.7554/eLife.28270.021
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Figure 4—figure supplement 1

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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

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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
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Figure 5—figure supplement 1

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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

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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

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PACSIN1 is a negative regulator of KCC2 expression and function.

(a) Example IV curves measuring E_GABA 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) E_GABA, (c) synaptic conductance, and (d) RMP from all experiments similar to the examples in a. (e) Summary of E_GABA 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

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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 name	UniProt ID	Spectral ratio	MaxP	ES	IS	P5	P50	pS940
KCC2b	Q91V14-2	349.0	1	X	X	X	X	X
KCC2a	Q91V14-1	203.8	1	X	X	X	X	X
PACSIN1	Q61644	136.0	1	X	X	X	X	X
SLC44A1	Q6X893	54.0	1				X
ATP2B4	Q6Q477	44.0	1	X			X
KIF21B	Q9QXL1	41.0	1			X
VCP	Q01853	24.0	1	X			X
GLS	D3Z7P3	23.5	1			X	X
RAB11FIP5	Q8R361	22.0	1	X			X	X
SRCIN1	Q9QWI6	17.0	1	X			X	X
RNF8	Q8VC56	16.0	1				X
JAGN1	Q5XKN4	12.0	1				X
PPFIA3	P60469	11.0	1	X			X	X
SLC2A1	P17809	11.0	0.96				X
YWHAE	P62259	10.0	0.99	X	X	X	X	X
ACTN4	P57780	9.0	1	X			X
CRMP1	P97427	8.0	1	X		X	X	X
PITPNM2	Q6ZPQ6	8.0	1				X	X
ACO2	Q99KI0	7.0	1	X		X	X	X
COX4I1	P19783	6.0	0.96				X
SLC4A10	Q5DTL9	6.0	1	X	X		X
USP24	B1AY13	6.0	1			X
YWHAG	P61982	6.0	0.96	X	X		X	X
C1QB	P14106	5.0	0.89				X	X
CCT6A	P80317	5.0	0.99	X		X
CPSF6	Q6NVF9	5.0	0.99				X
DDX5	Q61656	5.0	0.99	X		X
DYNC1LI1	Q8R1Q8	5.0	0.78	X		X	X
SEC23A	Q01405	5.0	0.99	X	X	X
SLC8A2	Q8K596	5.0	0.99	X			X
TRIM3	Q9R1R2	5.0	0.78	X	X	X	X
CANX	P35564	4.5	1	X	X	X	X
ATP5H	Q9DCX2	4.0	0.81				X
ATP6V1E1	P50518	4.0	0.78	X		X	X
DDX17	Q501J6	4.0	0.96			X
DNAJA3	Q99M87	4.0	0.78	X			X	X
DPYSL3	Q62188	4.0	0.96			X
EWSR1	Q61545	4.0	1			X	X	X
MAG	P20917	4.0	0.96	X	X		X
MDH2	P08249	4.0	0.96	X			X
RTCB	Q99LF4	4.0	0.98			X	X
SNRPA	Q62189	4.0	0.96			X
STOML2	Q99JB2	4.0	0.96	X		X
STRN3	Q9ERG2	4.0	0.96				X
YWHAQ	P68254	4.0	0.78	X			X
INA	P46660	3.6	1	X			X
ATP6V1F	Q9D1K2	3.5	0.95			X	X
DCTN2	Q99KJ8	3.5	1	X		X	X
CD47	Q61735	3.2	0.9		X		X
ACOX3	Q9EPL9	3.0	0.78				X
AP2B1	Q9DBG3	3.0	1	X	X		X
APBA1	B2RUJ5	3.0	0.78			X
ATP2A2	O55143	3.0	0.81	X			X	X
CMPK1	Q9DBP5	3.0	0.78	X		X
CRTC1	Q68ED7	3.0	0.78				X
CTNNA2	Q61301	3.0	0.78	X		X
FLII	Q9JJ28	3.0	0.78				X	X
FXYD7	P59648	3.0	0.78				X
GPM6A	P35802	3.0	0.93	X			X	X
GRID2	Q61625	3.0	0.78	X			X
NARS	Q8BP47	3.0	0.78				X
NDUFA2	Q9CQ75	3.0	1				X
NUDT21	Q9CQF3	3.0	0.78				X
PPP1CA	P62137	3.0	0.78	X	X	X
PPP2CA	P63330	3.0	0.78	X		X
PRRT2	E9PUL5	3.0	0.78		X		X	X
SFPQ	Q8VIJ6	3.0	1	X		X	X
SIRT2	Q8VDQ8	3.0	0.89				X	X
SLC1A2	P43006	3.0	0.69	X			X	X
TCP1	P11983	3.0	0.78	X		X
TRIO	Q0KL02	3.0	0.78	X		X
PTN	P63089	2.7	1			X
RAB2A	P53994	2.7	0.84	X			X
NDUFS5	Q99LY9	2.5	0.82				X
CCT2	P80314	2.5	1	X		X
DNM1	P39053	2.5	1	X			X
SLC25A11	Q9CR62	2.5	0.93	X	X		X
DDX1	Q91VR5	2.4	0.89	X		X	X
NEDD4L	Q8CFI0	2.4	0.91	X	X	X	X
SYNGR3	Q8R191	2.4	1	X			X	X
DLD*	O08749	2.3	0.44	X		X	X	X
SNAP25	P60879	2.3	0.65	X		X	X	X
DDX3X	Q62167	2.3	0.79	X		X	X	X
CAMK2G	Q923T9	2.3	1	X			X
FASN	P19096	2.3	1	X	X	X
PKM	P52480	2.2	0.85				X
NDUFA9	Q9DC69	2.1	0.96	X			X
BASP1	Q91XV3	2.1	0.72	X			X	X
CKB	Q04447	2.0	0.64	X			X
COX6C	Q9CPQ1	2.0	0.89				X
CSNK2A1	Q60737	2.0	0.84	X		X	X
DHX9*	O70133	2.0	0.44			X	X
DPYSL2	O08553	2.0	0.97	X		X	X	X
EDC4	Q3UJB9	2.0	1				X
FUS	P56959	2.0	0.94	X		X	X	X
KCNAB2	P62482	2.0	0.92	X			X
NDUFA8	Q9DCJ5	2.0	0.96	X			X
NDUFS8	Q8K3J1	2.0	1				X
PDIA6	Q922R8	2.0	0.89	X		X
SFXN3*	Q91V61	2.0	0.44	X			X
SLC25A22	Q9D6M3	2.0	0.89	X			X
STMN2	P55821	2.0	1			X
TNR	Q8BYI9	2.0	1	X	X		X
TUBB4B	P68372	1.9	0.55				X	X
ATP5C1	Q91VR2	1.9	0.6	X			X
PPIA	P17742	1.8	0.74			X	X
CKMT1	P30275	1.8	0.86	X		X	X	X
COX5A	P12787	1.8	0.54				X
C1QC*	Q02105	1.8	0.43				X	X
NDUFS1	Q91VD9	1.8	0.88	X	X		X
WWP1	Q8BZZ3	1.8	0.88				X
ATP5B	P56480	1.7	0.38	X		X	X	X
CCT5	P80316	1.7	0.81	X		X	X
DCLK1	Q9JLM8	1.7	0.96	X	X	X
SLC25A3	Q8VEM8	1.7	1	X	X		X
SPTBN1	Q62261	1.7	0.7			X	X
TUBB3	Q9ERD7	1.6	0.73			X	X	X
CAMK2D	Q6PHZ2	1.6	0.82	X		X	X
ATP6V0A1	Q9Z1G4	1.5	0.92	X			X
CFL1*	P18760	1.5	0.44	X		X	X
ATP1A2*	Q6PIE5	1.5	0.04	X		X	X	X
ADGRL2	Q8JZZ7	1.5	0.89			X
BSN	O88737	1.5	0.72	X			X
DBT	P53395	1.5	0.86	X		X	X
GTF2I	Q9ESZ8	1.5	0.89				X
HELB	Q6NVF4	1.5	0.89				X
HK1	P17710	1.5	0.89	X			X
HMCN2	A2AJ76	1.5	0.89				X
LGI3	Q8K406	1.5	0.89				X
PC	Q05920	1.5	0.89				X
RAB3IP	Q68EF0	1.5	0.89				X
UQCR11	Q9CPX8	1.5	0.89				X
ATP1A1	Q8VDN2	1.5	0.67	X	X		X	X
ATP5O	Q9DB20	1.5	0.71			X	X
NDUFA4	Q62425	1.4	0.51	X			X	X
ATP6V0D1	P51863	1.4	0.53	X			X
ACAT1*	Q8QZT1	1.4	0.43	X		X	X
ATP2B2*	Q9R0K7	1.4	0.47	X			X
TUBB4A	Q9D6F9	1.4	0.64			X	X	X
GNB1	P62874	1.4	0.58	X	X	X	X	X
C1QA	P98086	1.4	0.36			X	X
NDUFA10	Q99LC3	1.4	0.65	X			X
RAP2B*	P61226	1.3	0.42		X		X
SYT1	P46096	1.3	0.65	X			X	X
SLC1A3	P56564	1.3	0.9		X	X	X
CAPRIN1	Q60865	1.3	0.56			X	X
YWHAZ*	P63101	1.3	0.44	X	X	X	X	X
ATP1A3*	Q6PIC6	1.3	0	X		X	X	X
STX1B*	P61264	1.3	0.45	X	X		X	X
NEFM	P08553	1.2	0.78	X			X
DLST	Q9D2G2	1.2	0.72	X		X