Kainate Receptors Coexist in a Functional Complex with KCC2 and Regulate Chloride Homeostasis in...

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Kainate Receptors Coexist in a FunctionalComplex with KCC2 and Regulate ChlorideHomeostasis in Hippocampal NeuronsVivek Mahadevan,1 Jessica C. Pressey,1 Brooke A. Acton,1 Pavel Uvarov,5 Michelle Y. Huang,1 Jonah Chevrier,1

Andrew Puchalski,1 Caiwei M. Li,1 Evgueni A. Ivakine,2 Matti S. Airaksinen,5 Eric Delpire,3 Roderick R. McInnes,2,4

and Melanie A. Woodin1,*1Department of Cell & Systems Biology, University of Toronto, Toronto, ON M5S 3G5, Canada2Hospital for Sick Children Research Institute, Toronto, ON M5G 1X8, Canada3Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA4Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada5Institute of Biomedicine, Anatomy, University of Helsinki, 00014 Helsinki, Finland

*Correspondence: [email protected]://dx.doi.org/10.1016/j.celrep.2014.05.022

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY

KCC2 is the neuron-specific K+-Cl� cotransporterrequired for maintaining low intracellular Cl�, whichis essential for fast inhibitory synaptic transmissionin the mature CNS. Despite the requirement ofKCC2 for inhibitory synaptic transmission, under-standing of the cellular mechanisms that regulateKCC2 expression and function is rudimentary. Weexamined KCC2 in its native protein complex in vivoto identify key KCC2-interacting partners thatregulate KCC2 function. Using blue native-polyacryl-amide gel electrophoresis (BN-PAGE), we deter-mined that native KCC2 exists in a macromolecularcomplex with kainate-type glutamate receptors(KARs). We found that KAR subunits are requiredfor KCC2 oligomerization and surface expression.In accordance with this finding, acute and chronicgenetic deletion of KARs decreased KCC2 functionand weakened synaptic inhibition in hippocampalneurons. Our results reveal KARs as regulators ofKCC2, significantly advancing our growing under-standing of the tight interplay between excitationand inhibition.

INTRODUCTION

Hyperpolarizing GABAergic synaptic transmission in the mature

CNS depends upon a low concentration of intracellular Cl� [Cl�]iKCC2 is the neuron-specificmember of the K+-Cl� cotransporter

gene family that primarily extrudes Cl� from neurons, making it

essential for inhibitory synaptic transmission (Acton et al.,

2012; Blaesse et al., 2009; Rivera et al., 1999). Physiological

levels of neuronal activity can regulate KCC2 in a Ca2+-depen-

dent manner to induce inhibitory synaptic plasticity, which plays

a key role in the delicate balance between inhibition and excita-

tion (Fiumelli and Woodin, 2007; Lamsa et al., 2010; Woodin

1762 Cell Reports 7, 1762–1770, June 26, 2014 ª2014 The Authors

et al., 2003). However, aberrant KCC2 regulation results in

increased neuronal Cl� and contributes toward the pathophysi-

ology of numerous neurological disorders including epilepsy,

autism, and neuropathic pain (Coull et al., 2005; Kahle et al.,

2008; Tyzio et al., 2014; Woo et al., 2002).

KCC2 membrane expression and function are regulated by

multiple posttranslational mechanisms, including alterations in

phosphorylation state, oligomerization, association with lipid

rafts, and cleavage by proteases (Blaesse et al., 2006; Lee

et al., 2011; Puskarjov et al., 2012; Rinehart et al., 2009; Wata-

nabe et al., 2009). Recently, we made an important addition to

this list ofmechanisms that regulate KCC2 function by identifying

a KCC2-interacting protein termed Neto2 (Ivakine et al., 2013).

We found that Neto2 is required to maintain KCC2 abundance

in neurons and for efficient KCC2-mediated Cl� transport.

Thus, the KCC2-Neto2 interaction is vital for normal synaptic in-

hibition in mature neurons.

Neto2 is a CUB domain containing transmembrane protein

that also acts as an auxiliary subunit of native kainate-type gluta-

mate receptors (KARs). Neto2 regulates both the kinetics and

synaptic localization of KAR subunits (Copits et al., 2011; Tang

et al., 2012; Wyeth et al., 2014; Zhang et al., 2009). KARs are

unique ionotropic glutamate receptors that perform multiple

functions during synaptic transmission and plasticity (Lerma

and Marques, 2013). They regulate GABAergic release from

presynaptic terminals (Rodrıguez-Moreno et al., 1997), mediate

slow excitatory currents postsynaptically (Castillo et al., 1997),

and are involved in mossy fiber-pyramidal neuron long-term

potentiation in the CA3 area (Contractor et al., 2001).

Our identification of the Neto2-KCC2 interaction, coupled with

the previous demonstrations that Neto2 is an auxiliary subunit of

KARs, led us to ask whether KCC2 and KARs coexist in a macro-

molecular complex. In particular, we examined the role of GluK2

subunits that were previously shown to interact with Neto2

(Copits et al., 2011; Tang et al., 2011; Zhang et al., 2009). In

this study, we have made a surprising discovery that native

oligomeric KCC2 coexists in an ensemble with the GluK2 KAR

subunit in the CNS. Moreover, we determined that KARs

are required to maintain both KCC2 oligomerization and the

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Figure 1. KCC2 Interacts with GluK2 KARs

in Mouse Brain and in Heterologous Cells

(A) Native KCC2 complexes fromC12E9-solubilized

whole-brain membrane fractions immunoprecipi-

tated with anti-KCC2 and immunoblotted with the

antibodies indicated at right (KCC2, GluK2/3,

Neto2). Representative example of three inde-

pendent replicates. IP, immunoprecipitate; I, input

fraction (1% of IP); U. unbound fraction (1% of IP);

O. oligomer; M. monomer; also see Figure S1A.

(B) (Bi) Coimmunoprecipitation experiments per-

formed in HEK293 cells transfected with KCC2 and

KAR subunits, solubilized in RIPA buffer, immu-

noprecipitated with anti-KCC2, and immuno-

blotted with the antibodies indicated at right

(KCC2, GluK2/3, myc); also see Figures S1B and

S1C. Representative example of three to four in-

dependent biological replicates (Bii) Quantitation

of the bound fractions to KCC2 was performed by

measuring the band intensity of the immunopre-

cipitated fraction compared with total input (10%)

using ImageJ software.

(C) Confocal images of DIV 12-14 cultured mouse

hippocampal neurons immunostained for endog-

enous KCC2 (left, green) and GluK1/2 (middle,

red), demonstrating that the two proteins are co-

localized (right, yellow). Representative of confocal

images obtained from 26 neurons over four inde-

pendent experiments performed using eight cov-

erslips. (Scale bars, 10 mm.) Bottom inset is a

magnification from the primary dendrite indicated

in the box.

All summary figures represent mean ± SEM. *p <

0.05, **p < 0.01, ***p < 0.001.

expression of this transporter in the membrane. When we per-

formed an electrophysiological characterization of KCC2 func-

tion following KAR subunit disruption, we found neurons had

a depolarized reversal potential for GABA (EGABA). Hence, our

findings represent a regulation of KCC2 function and fast synap-

tic inhibition by components of excitatory transmission.

RESULTS

KCC2 and GluK2 KARs Interact In Vivo and In VitroWe have recently discovered that KCC2 binds to the single-pass

CUB domain protein Neto2, and that this interaction is required

for efficient Cl� extrusion in hippocampal neurons (Ivakine

et al., 2013). Several groups have previously established that

Neto2 is a critical auxiliary subunit of native KARs, including

GluK2 (Copits et al., 2011; Tang et al., 2011; Wyeth et al.,

2014; Zhang et al., 2009). This led us to hypothesize that KARs

Cell Reports 7, 1762–177

might be a putative candidate that

could interact with KCC2. In order to

determine whether KCC2 interacts

with KAR subunits in vivo, we performed

a coimmunoprecipitation assay from

whole-brain native membrane prepara-

tions. We found that anti-KCC2 anti-

bodies coimmunoprecipitated GluK2/3

primarily from wild-type mice in comparison to KCC2b+/�

mice, indicating the existence of a KCC2-KAR complex in vivo

(Figures 1A and S1A; n = 3). To determine whether KCC2 can

interact with KARs independent of exogenous Neto2, we per-

formed coimmunoprecipitation experiments in HEK293 cells

transfected with KCC2 and KAR subunits alone. In this assay,

we found that KCC2 could coimmunoprecipitate GluK2, but

not GluK1 (Figures 1B and S1B; n = 4). We also performed the

experiment in the reverse direction and found that GluK2, but

not GluK1, could also robustly coimmunoprecipitate KCC2 (Fig-

ure S1C; n = 3). Based on the interaction of KCC2 and GluK2 in

these coimmunoprecipitation experiments, we hypothesized

that these two proteins would colocalize in neurons. We tested

this hypothesis by performing immunofluorescent staining of

endogenous proteins using antibodies specific for KCC2

and GluK1/2 in cultured hippocampal neurons, followed by

quantitative colocalization. Hippocampal neurons showed

0, June 26, 2014 ª2014 The Authors 1763

Figure 2. Native KCC2 and KARs Exist in the

Same Oligomeric Complex

(A) One-dimensional BN-PAGE separation of

native KCC2 and GluK2/3 complexes from P30

mouse brain solubilized with Digitonin or C12E9; gel

separations were immunoblotted with the anti-

bodies indicated above (KCC2, GluK2/3). This blot

is a representative example of three independent

biological replicates. Native KCC2 is present as a

putative tetramer (blue arrow head), and as higher-

order protein complexes (green arrow heads).

Native GluK2/3 migrates in a similar molecular

weight range as native KCC2 (dotted yellow lines);

also see Figure S2A.

(B) (Bi) Antibody-shift assay followed by two-

dimensional BN-PAGE separation using C12E9-

solubilized whole-brain membrane fractions,

incubated with antibodies for HA or GluK2/3;

samples resolved first in a 5% 1D-BN-PAGE, and

individual lanes from the first dimension were

separated in a 6% 2D-SDS-PAGE); gel separa-

tions were immunoblotted with anti GluK2/3

antibody. (Bii and Biii) Similar to (Bi), but using

digitonin-solubilized hippocampal membrane

fractions and antibodies targeted to immunoglob-

ulin G or GluK2/3. Samples resolved in a 4% 1D-

BN-PAGE and 6% SDS-PAGE. Gel separations

were immunoblotted with KCC2 and Neto2 anti-

bodies respectively. Bottom panels in Bi, Bii, Biii

represent densitometric profiles and area under

the densitogram to indicate similar loading; red

arrowheads denote antibody-induced shifts in the

proteins indicated. Representative example of

three independent biological replicates; also see

Figures S2B–S2D.

immunofluorescence for both endogenous KCC2 and KARs,

with a partial colocalization of these two proteins (Figure 1C;

n = 26). We then performed an intensity correlation analysis to

quantitate the colocalization and calculated a Pearson’s correla-

tion coefficient of 0.61 ± 0.03 between the GluK1/2 and KCC2

immunofluorescent signals, indicating �60% colocalization of

these proteins. Thus, we have discovered a surprising protein

interaction between KCC2 and the predominant kainate receptor

subunit, GluK2.

Native KCC2 Exists in a Hetero-Oligomeric Ensemblewith Native KARsFunctional KCC2 predominantly exists as an oligomer in the

mature brain (Blaesse et al., 2006; Ivakine et al., 2013; Uvarov

et al., 2009). Consistently, we observed that native KCC2 exist

as a putative tetramer and in higher-order complexes above

400 kDa (Figures 2A and S2A; n = 3), in P30 whole-brain mem-

brane lysates using a one-dimensional blue native polyacryl-

amide gel electrophoresis (1D-BN-PAGE). Similar to native

KCC2, we also observed higher-order complexes of comparable

molecular weights that contained native GluK2/3 (Figures 2A and

S2A). Next, to determine whether native KCC2 exists in an

ensemble with KAR subunits, we employed an antibody-shift

assay coupled with two-dimensional blue native polyacrylamide


gel electrophoresis (2D-BN-PAGE). The 2D-BN-PAGE strategy

has been previously employed to examine the native assemblies

of AMPA receptor multimeric complex (Schwenk et al., 2009).

Using this approach, we first verified that the addition of

the GluK2/3 antibody could shift GluK2/3 to higher molecular

weights (Figure 2Bi; n = 3). We observed that this antibody-

induced shift in GluK2/3 also shifted a population of native

KCC2 in hippocampal preparations (Figure 2Bii; n = 3). Using

the same experimental strategy, we found that KCC2 antibodies

could also shift a population of GluK2/3 in hippocampal prepara-

tions (Figure S2C; n = 3), a finding that we also observed

in digitonin-solubilized cortical membrane preparations (Fig-

ure S2D; n = 2). As a positive control for this assay, we probed

for Neto2, because this protein interacts with both KCC2

and GluK2 (Ivakine et al., 2013; Tang et al., 2011; Zhang et al.,

2009). As expected, we found that Neto2 could be shifted

with both GluK2/3 (Figure 2Biii) and KCC2 antibodies (Fig-

ure S2D). We confirmed the specificity of these interactions in

this assay by repeating the experiments using antibodies for

the transferrin receptor and observed that antibodies to this

receptor did not shift GluK2/3 (Figure S2B). Thus, we established

that functional oligomeric KCC2 coexists in a hetero-oligomeric

complex with the predominant KAR subunit GluK2 in hippocam-

pus and cortex.

Figure 3. GluK1/2-Null Hippocampal

Neurons Have an Increased Monomeric:

Oligomeric KCC2 Ratio

(A) Representative immunoblots of C12E9-solubi-

lized native lysates prepared in the presence of

25 mM iodoacetamide to prevent the formation of

nonspecific disulphide bonds between KCC2

monomers during membrane extraction, from

wild-type and GluK1/2-null hippocampi; resolved

in a standard 6% SDS-PAGE; immunoblotted with

the antibodies indicated at left (KCC2, NKCC1,

GluK2/3, ATP1A3, Tuj1). Also see Figure S3Ai.

(B) Samples obtained from the same preparation

as in (A) were resolved in the absence of DTT

in nondenaturing PFO-PAGE conditions. Red

arrowhead indicates the oligomeric KCC2 band

migrating above �400 kDa that is predominantly

reduced in GluK1/2�/� lysates; blots shown in (A)

and (B) are representative of three independent

biological replicates, see Figures S3Aii and S4A.

(C) Summary figures showing levels of (Ci) KCC2

monomers, (Cii) KCC2 oligomers, (Ciii) total KCC2,

and (Civ) monomer:oligomer KCC2 ratio in GluK1/

2-null hippocampal homogenates relative to that of

wild-type.

(D) (Di) Example confocal microscopic immuno-

fluorescent images of cultured hippocampal neu-

rons from wild-type and GluK1/2-null mice stained

with anti-KCC2 antibody (red; n = 49 neurons).

(Scale bars, 24 mm.) (Dii) Summary of the average

fluorescence intensity of somatic KCC2 in wild-

type and GluK1/2-null neurons.


0.05, **p < 0.01, ***p < 0.001.

KARs Regulate the Assembly or Stability of Native KCC2OligomersIf oligomeric KCC2 exists in a complex with GluK2, and oligo-

meric KCC2 is the functional form of this transporter, this raises

the possibility that KARs may play an important role in the regu-

lation of KCC2 oligomers. To test this possibility, we examined

the ratio of KCC2 monomers to oligomers in native membranes

prepared from wild-type and GluK1/2-null hippocampal lysates

under conditions preventing the formation of nonspecific disul-

phide bonds (Uvarov et al., 2009). In an SDS-PAGE, we observed

an increase in monomeric KCC2 and a decrease in oligomeric

KCC2 in GluK1/2-null hippocampi (Figures 3A and S3Ai; n = 3).

We also examined the levels of the neuron-specific pump

ATP1A3 and another KCC2-family member NKCC1, and

observed no significant difference between wild-type and

GluK1/2-null lysates (Figure 3A). We reasoned that the dena-

turing gel running conditions of SDS-PAGE could impede a

robust quantification of KCC2 oligomeric levels, so we subse-


quently chose to resolve changes in the

monomer:oligomer ratio using a previ-

ously established nondenaturing PFO-

PAGE (Uvarov et al., 2009). Using these

conditions, we observed a 2-fold increase

in monomeric KCC2 in GluK1/2-null

hippocampi (Figures 3B, 3Ci, and S3Aii;

n = 3) as expected from the previous SDS-PAGE result. In addi-

tion, we also observed a significant decrease in oligomeric KCC2

levels above �400 kDa in GluK1/2-null hippocampi compared

with wild-type levels (Figures 3B, 3Cii, and S3Aii), and no signifi-

cant change in total KCC2 levels under the same conditions (Fig-

ure 3Ciii). We verified that there is no change in total KCC2 levels

by an additional standard approach by preparing the samples in

the absence of iodoacetamide, and resolved them under strong

denaturing conditions, indicating that there is no net change in

total KCC2 levels (Figure S3Aiii; n = 3). Additionally, we verified

that the increases inmonomeric KCC2 levels were not accompa-

nied by changes in KCC2 gene expression, by examining the

relative KCC2 mRNA abundance using quantitative real-time

PCR. We found no significant differences between wild-type

and GluK1/2-null hippocampi prepared from postnatal day 30

mice (Figure S3B; n = 3). Put together, these results demonstrate

that the presence of GluK1/2 determines the monomer:oligomer

ratio of KCC2 (Figure 3B and 3Civ) at the posttranscriptional


Figure 4. GluK1/2-Null Neurons Have

Deceased KCC2 Expressed at the Surface

(A) (Ai) Representative immunoblots of KCC2

monomers and oligomers from the surface and

internal fraction of wild-type and GluK1/2-null

neurons. The first two lanes correspond to bio-

tinylated surface proteins (50 mg) recovered from

the neutravidin beads. The last two lanes corre-

spond to unbiotinylated internal proteins (5 mg)

recovered from the supernatant. (Aii–v) Summary

figures showing levels of surface KCC2 mono-

mers, surface KCC2 oligomers, total surface KCC2

(normalized to surface levels of neuron-specific

ATP1A3), and internal KCC2 (normalized to b-actin

levels) in GluK1/2-null homogenates relative to that

of wild-type (n = 5).

(B) (Bi) Example confocal microscopic immuno-

fluorescent images from a single confocal plane of

live cultured hippocampal neurons overexpressing

KCC2-HA from wild-type and GluK1/2-null mice

(also see Figure S4B). Neuronal transfection is

indicated by GFP fluorescence, KCC2-HA is indi-

cated by red fluorescent signal. Summary of the

(Bii) average number of anti-HA puncta/neuron,

(Biii) percentage of anti-HA puncta proximal to the

surface (Biv) mean puncta size (surface area, mm2),

and (Bv) puncta intensity (in arbitrary units x1000) in

wild-type and GluK1/2-null neurons. (Bvi) Total

immunofluorescence of live imaging of wild-type

and GluK1/2-null neurons. n = 23 neurons.


0.05, **p < 0.01, ***p < 0.001.

level. Moreover, by showing that loss of KAR subunits induces a

significant reduction in KCC2 oligomers, particularly above

�400 kDa, we further strengthen our two major claims: (1) the

existence of a KAR:KCC2 hetero-oligomeric complex in the hip-

pocampus, and (2) KARs promote the assembly or the stability of

KCC2 oligomers within the complex.

KARs Maintain KCC2 Surface Expression in NeuronsIt has been previously demonstrated by several groups that

immature neurons are characterized by predominantly mono-

meric KCC2 that exists intracellularly (reviewed in Chamma

et al., 2012). Based on these previous demonstrations, we exam-

ined whether there were differences in the expression patterns of

KCC2 between wild-type and GluK1/2�/� neurons. We first


examined total KCC2 levels by per-

forming immunofluorescent staining of

cultured hippocampal neurons with anti-

KCC2 antibody. We found a significant

increase in endogenous KCC2 immuno-

reactivity in the soma of GluK1/2�/�

neurons compared to wild-type neurons

(wild-type, n = 47; GluK1/2�/�, n = 49;

p < 0.001; Figure 3D). However, for

KCC2 to be functional it needs to be

expressed in the membrane. Next, we

examined the membrane expression

pattern of KCC2 by performing a surface biotinylation assay at

4�C. We found a significant decrease in monomeric (p =

0.029), oligomeric (p = 0.029), and total (p = 0.008) KCC2 levels

in the surface of GluK1/2�/� neurons compared to wild-type

neurons, with a corresponding increase in internal monomeric

KCC2 (p = 0.008; n = 5; Figure 4A). To visualize KCC2 expres-

sion, we performed live immunofluorescence of KCC2 contain-

ing an extracellular HA tag under nonpermeabilizing conditions

at 37�C. We have previously demonstrated that this KCC2-HA

chimeric protein traffics to the membrane and is functional (Ac-

ton et al., 2012). We found that in GluK1/2�/� neurons there

was a significant decrease the number of anti-HA puncta (Fig-

ure 4Bii; n = 23), with no significant differences in either the

size or intensity of the puncta (Figures 4Biv and 4Bv; n = 23),

Figure 5. Both Genetic Deletion and Acute

Knockdown of GluK2 KAR Subunits Depo-

larizes EGABA

(A) Example IV curves measuring EGABA from

cultured hippocampal neurons from wild-type (left)

and GluK1/2�/� (middle) mice. Summary of EGABA

obtained from all similar IV curves (right).

(B) Example IV curves measuring EGABA from

cultured hippocampal neurons from wild-type

transfected with scrambled shRNA (left) and

shRNA for GluK2 (middle). Summary of EGABA ob-

tained from all similar IV curves.

(C) Summary of EGABA recordings performed in the

absence (control) and presence of the GluK2/5-

KAR inhibitor (5 mM UBP 310). Solid circles are

individual EGABA measurements, open triangles

represent the mean ± SEM. Insets: are raw voltage

clamp traces from example recordings.

Scale bars, 20 pA, 50 ms. Arrow indicates onset of

GABA puff. Dashed vertical lines indicate where

the current amplitudes were obtained for the

generation of the IV curves. All summary figures

represent mean ± SEM. *p < 0.05, **p < 0.01, ***p <

0.001.

indicating an overall decrease in the number of KCC2-HA

puncta, with no difference in the puncta characteristics them-

selves. We then analyzed where the existing puncta were

located and found that there was a significant decrease in the

KCC2-HA puncta that were proximal to the membrane in

GluK1/2�/� neurons (Figure 4Biii and S3; n = 23). We made two

important observations from these biotinylation and immuno-

staining experiments: (1) overall there is an increased total

KCC2 immunoreactivity in soma of GluK1/2�/� neurons (Fig-

ure 3D); and (2) GluK1/2�/� neurons have a decreased mem-

brane expression (Figure 4A). Thus, in addition to promoting

and/or stabilizing KCC2 oligomers, the presence of KAR sub-

units GluK1/2 also promotes and/or stabilizes surface KCC2

levels.

Genetic Deletion and Acute Silencing of KAR SubunitsResult in Depolarized EGABA

Because we observed a decrease in both oligomeric KCC2 and

surface KCC2 in GluK1/2-null neurons, we hypothesized that

these neurons would have aberrant KCC2-mediated Cl� homeo-


stasis. To test this hypothesis, we mea-

sured KCC2 activity by recording the

reversal potential for GABA (EGABA) using

gramicidin-perforated patch clamp re-

cordings. We found that EGABA was depo-

larized in cultured GluK1/2�/� hippocam-

pal neurons (�65.2 ± 1.9 mV; n = 10)

compared to wild-type neurons (�78.6 ±

3.5; n = 7; p = 0.003; Figure 5A), with no

significant difference in either synaptic

conductance (p = 0.5) or maximum cur-

rent amplitude (p = 0.6; Table S2). We

then took a two-step approach to rule

out the possibility that the depolarization

of EGABA in GluK1/2�/� neurons was due to differences in activity

levels between the genotypes. First, we compared the sponta-

neous activity levels using Ca2+ imaging and found no differ-

ences between cultured hippocampal neurons prepared from

wild-type (C57/Bl6 and 129SVE) and GluK1/2�/� mice (Fig-

ure S5A). Second, we used small hairpin RNA (shRNA) to acutely

silence GluK2 in cultured hippocampal neurons; we verified

GluK2 shRNA was effective at silencing GluK2 but not KCC2 us-

ing an in vitro assay (Figure S5B). We found that knocking down

GluK2 in wild-type neurons (C57/Bl6) depolarized EGABA by

15.41 mV compared to neurons transfected with scrambled

shRNA (Figures 5B and S5B; n = 11). Knocking down GluK2 in

129SVE wild-type neurons also significantly depolarized EGABA

(n = 11; p < 0.05). Similar to our results from cultured GluK1/

2�/� hippocampal neurons above, we found no significant differ-

ence in either synaptic conductance (p = 0.6) or maximum cur-

rent amplitude (p = 0.1) for either genotype (Table S2). Last, we

considered the possibility that the depolarization in EGABA we re-

corded following the genetic deletion and acute silencing of KAR

subunits was not due to the loss of the protein, but rather was


due to the loss of the GluK2-KAR current. We tested this possi-

bility by recording EGABA in wild-type neurons in the presence

and absence of the GluK2/5-KAR antagonist UBP 310 (5 mM)

(Pinheiro et al., 2013).We found no significant difference in EGABA

following this pharmacological blockade of these receptors (n =

5; p = 0.159; Figure 5C), allowing us to conclude that it is the

protein interaction between GluK2-KARs and KCC2 that is

required to maintain a high KCC2 function.

DISCUSSION

Overall, we have three compelling lines of evidence supporting

the conclusion that KCC2 and KAR subunits coexist in a

complex: (1) in vivo and in vitro coimmunoprecipitation, (2)

antibody-shift assay coupled with 2D-BN-PAGE, and (3) immu-

nofluorescence. Thus, the key K+-Cl� cotransporter required

for fast synaptic inhibition binds to the predominant KAR-type

ionotropic glutamate receptor subunit GluK2 in multiple brain re-

gions including hippocampus. Furthermore, we find that the

GluK2:KCC2 interaction occurs predominantly with oligomeric

KCC2, and the importance of this preferential binding is to main-

tain the functional oligomeric KCC2 complex. Along with our

evidence that both genetic deletion and acute knockdown of

GluK2 result in aberrant KCC2-dependent neuronal Cl� extru-

sion, we have identified kainate receptors as an unexpected

player during neuronal Cl� homeostasis.

Functional KCC2 Exists as a Hetero-OligomericComplex with KARsSeveral studies have established that functional KCC2 predom-

inantly exists as oligomers in mature neurons (Blaesse et al.,

2006; Ivakine et al., 2013; Uvarov et al., 2009; Watanabe et al.,

2009). Here, we report using BN-PAGE that the majority of olig-

omeric KCC2 in mature brain migrates above 400 kDa. The key

finding of this study is that GluK2 is a member of this KCC2-het-

eromeric complex. This finding raises an important question: is

this complex exclusive to KCC2 and GluK2, or do these com-

plexes also include Neto2 and other proteins? This question is

relevant because we recently identified that Neto2 associates

with oligomeric KCC2 (Ivakine et al., 2013). Our current data

demonstrate that, whereas KCC2 and Neto2 interact, Neto2 is

not required for the GluK2:KCC2 interaction because GluK2

and KCC2 can interact in the absence of exogenous Neto2 in

heterologous cells. Despite the fact that Neto2 is not required

for the GluK2:KCC2 interaction, this does not discount the pos-

sibility that these three proteins commonly exist in a heteromeric

complex. Moreover, there is another reason to believe that addi-

tional proteins may also coexist in this heteromeric complex. For

example, the 4.1N protein has been identified as binding part-

ners of both KCC2 (Li et al., 2007) and more recently GluK2

(Copits and Swanson, 2013). Because the 4.1 family of FERM

domain proteins are abundant scaffolds between membrane

and cytoskeletal proteins (Baines et al., 2013), it is possible to

speculate that the GluK2:KCC2 interaction we have identified

could be mediated/stabilized by neuronal FERM domain pro-

teins. Interestingly, while this manuscript was in revision another

group discovered that the dwell time of KCC2 in the vicinity of

excitatory synapses is determined by its interactions with 4.1N


(Chamma et al., 2013), further strengthening our claims about

the putative participation of the 4.1N protein within the KCC2:

KAR hetero-oligomeric complex.

KCC2 Oligomerization and Surface Expression Dependon GluK2-KARsWe found that GluK1/2-null hippocampal neurons have a

decrease in KCC2 oligomers, particularly above 400 kDa. This

suggests that GluK2 plays a role in either the formation of

KCC2 oligomers and/or regulates their stability, but how might

this happen? The answer may lie in the fact that GluK2-null

hippocampi have an �50% reduction in Neto2 protein levels

(Figures 4A and S4A), and we know that Neto2 is required for

KCC2 oligomerization and the efficacy of KCC2 transport (Iva-

kine et al., 2013). Does this mean that GluK2 regulates KCC2

via only Neto2, or can it directly regulate KCC2 function? Our

current results suggest that GluK2 can also regulate KCC2 inde-

pendently of Neto2. We found that in GluK1/2�/� neurons there

was an increase in KCC2 monomers and a decrease in KCC2

oligomers, with no net change in total KCC2 levels. This is in

contrast to our previous finding that Neto2-null neurons have

an overall decrease in both monomeric and oligomeric KCC2,

which results in a total decrease in KCC2 protein levels (Ivakine

et al., 2013).

Results from our biotinylation experiments allowed us to

conclude that GluK1/2�/� neurons have a decrease in cell-sur-

face KCC2. Thus, in addition to promoting or stabilizing KCC2

oligomers GluK1/2-KARs also maintain and/or stabilize surface

KCC2 levels. We supported these findings by immunostaining

for KCC2; using standard fixed immunofluorescence, we found

an increase in somatic KCC2 levels, whereas live immunofluo-

rescence of KCC2-HA showed a decrease in the number of

anti-KCC2-HA puncta. Although anti-KCC2-HA puncta must

have been present on the surface at some point during the

experiment, we cannot conclude that our puncta analysis repre-

sents only membrane expressed protein. At the temperature

these experiments were performed (37�C), we would expect

some proportion of KCC2-HA would be endocytosed, especially

considering that KCC2 membrane turnover has been reporter to

be relatively high (Lee et al., 2010, but see also Puskarjov et al.,

2012). Thus, the KCC2 puncta we have quantified may represent

a combination of KCC2-HA in the membrane and endocytotic

vesicles. Thus, although we are able to conclude that the loss

of GluK2-KARs decreases KCC2 membrane expression, future

studies should address whether GluK2 also regulates KCC2

membrane turnover. Together, the increase in KCC2 monomers

and decrease in membrane expression suggests that GluK1/

2�/� neurons resemble immature neurons, which have an abun-

dance of cytoplasmic KCC2monomers (Gulyas et al., 2001). Our

electrophysiological data support this observation of an imma-

ture Cl� homeostasis phenotype, where the genetic deletion of

GluK1/2 is reminiscent of immature neurons with poor Cl� extru-

sion, suggesting that kainate receptors are an essential compo-

nent of mature neuronal Cl� homeostasis.

Conclusions and Future SignificanceThe significance of our findings are manifold, but most impor-

tantly: (1) KCC2 and KARs exist in the same macromolecular

complex, and (2) an ionotropic glutamate receptor can positively

regulate the function of the predominant neuronal Cl� cotrans-

porter KCC2. These findings have important implications for

both normal physiological functions of neuronal networks and

for pathophysiological conditions that result from dysfunction

of KARs and KCC2. At the physiological level, we have demon-

strated that both genetic deletion and acute knockdown of

GluK2 weakens synaptic inhibition, suggesting that the co-

existence of these proteins provides a nexus for the ongoing

maintenance of the excitatory-inhibitory balance. At the patho-

physiological levels, both KCC2 and KARs are strongly impli-

cated in neurological disorders, including neuropathic pain

(Bhangoo and Swanson, 2013), autism (Tyzio et al., 2014), and

epilepsy (Woo et al., 2002). This raises the possibility that the

disruption of the KCC2:KAR complex may underlie these neuro-

physiological disorders. Understanding the fundamental molec-

ular pathways that regulate the cell intrinsic excitation, inhibition

homeostasis is essential for designing of better therapeutic stra-

tegies for diseases.

EXPERIMENTAL PROCEDURES

Animals and Approvals

All experiments were performed in accordance with approval and guidelines

from the University of Toronto Animal Care Committee and the Canadian

Council on Animal Care. Animals of both sexes were used to prepare hippo-

campal cultures; all other experiments were performed on male mice. The

following animal species were used:

d Wild-type C57/Bl6 (Charles River Laboratories)

d Wild-type 129/SV (Charles River Laboratories)

d GluK1/2�/� maintained on a mixed 129SV/C57Bl6 background

d KCC2+/� maintained on a mixed 129SV/C57Bl6 background

Antibodies

See Table S1 for complete details for all antibodies used in this study.

Biochemistry and Molecular Biology

See Supplemental Experimental Procedures for coimmunoprecipitation anal-

ysis, PFO-PAGE, BN-PAGE, antibody-shift assay, PCR, surface biotinylation.

Hippocampal Cultures and Electrophysiology

Low-density cultures of dissociated mouse hippocampal neurons were

prepared as previously described (Acton et al., 2012). Experiments were

performed after 10–13 days in culture. Gramicidin perforated patch clamp re-

cordings were performed as previously described (Acton et al., 2012). See

Supplemental Experimental Procedures for details on culturing and electro-

physiology, shRNA, and neuronal transfection.

Immunostaining, Confocal Microscopy, Ca2+ Imaging

Live immunostaining was performed as described before (Acton et al., 2012).

See Supplemental Experimental Procedures for details on fixed and live immu-

nostaining, confocal microscopy, and Ca2+ imaging.

Statistics

Results are given as mean ± SEM. See Supplemental Experimental Proce-

dures for details on statistical tests used for individual figures.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

five figures, and two tables and can be found with this article online at http://

dx.doi.org/10.1016/j.celrep.2014.05.022.

C

AUTHOR CONTRIBUTIONS

V.M. and M.A.W. designed the study, performed experiments, analyzed data,

and wrote the paper. J.C.P., B.A.A., P.U., M.Y.H., J.C., A.P., and C.M.L. per-

formed experiments. V.M., E.A.I., M.S.A., E.D., R.R.M., and M.A.W. inter-

preted data, critically analyzed, and edited the manuscript.

ACKNOWLEDGMENTS

We thank Dr. Derek Bowie (McGill University) for the GluK2-GFP cDNA, Dr.

Sari Lauri (University of Helsinki) for the GluK1b-myc cDNA, Dr. David B.Mount

for KCC4 cDNA, anti-KCC4 antibody, and Dr. Chris McBain (NIH, Bethesda)

for the GluK1/2�/� mice. We thank Dr. Mike Salter (University of Toronto) for

helpful insight throughout the project. We thank Dr. Nivetha Ramachandran

and Dr. Sakthi Devi Moorthy for technical advice. In addition, we thank Ella

Czerwinska for excellent technical support. This study was supported the

following funding agencies: Canadian Institutes of Health Research (CIHR)

grant to M.A.W.; NIH grant (GM074771) to E.D.; The Academy of Finland

grants to P.U. and M.S.A.

Received: May 24, 2013

Revised: March 31, 2014

Accepted: May 9, 2014

Published: June 5, 2014

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SUPPLEMENTAL INFORMATION

Kainate receptors co-exist in a functional complex with KCC2 and regulate chloride

homeostasis in hippocampal neurons

Vivek Mahadevan, Jessica C. Pressey, Brooke A. Acton, Pavel Uvarov, Michelle Y. Huang,

Jonah Chevrier, Andrew Puchalski, Caiwei M. Li, Evgueni A. Ivakine, Matti S. Airaksinen, Eric

Delpire, Roderick R. McInnes, Melanie A. Woodin

Supplementary Figure 1, Related to Figure 1

Wildtype KCC2b+/-

O

M

1 2 3 4 5 6

I IP U I IP U

250

130 IP:

KC

C2

60

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KC

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GluK2

GluK1b-myc

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IP:

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+ - -

- + -

- - +

+ + -

+ - +

- - -

+ - -

- + -

- - +

+ + -

+ - +

c KCC2

GluK2-FLAG

GluK1b-myc

- + + + - - + - - - - +

KC

C2

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kDa

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2

ci

IgG

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KC

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1.0 0.72 0.48

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~kDa

cii

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BN-PAGE (1D)

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)

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aii

WT

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250

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20%

40%

60%

80%

100%

120%

Fig. Solubilisation

Sample

processing

Gel

running

S3ai

C12E9 buffer +

25mM IAA

SDS-SB, 100mM

DTT, 37*C

SDS-

PAGE

S3aii

C12E9 buffer +

25mM IAA

PFO-SB

(cold)

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S3aiii

RIPA buffer

without IAA

SDS-SB, 100mM

DTT, 100*C

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aiv


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0

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+

+

+

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KCC2

GluK2

β-actin

KCC4

myc

β-actin

KCC4

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Scrambled shRNA

GluK2 shRNA#3

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100

80

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scrambled

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β-actin

KCC2 and KARs Co-Exist in a Complex Mahadevan et al

Supplemental Material

7

Supplemental Figure 1 (Related to Figure 1): KCC2 interacts with GluK2 KARs in

the mouse brain and in heterologous cells. (a) Higher exposure of Fig. 1a showing reduced levels of KCC2 oligomers

immunoprecipitated in KCC2b+/-

lysate and decreased levels of Neto2 and GluK2/3 co-

precipitated (as indicated by the dotted line yellow boxes). (b) Same experiment as Fig.

1b, but the blots were stripped and re-probed with antibody raised against GluK1/2. This

is especially important because the GluK2/3 antibody that was used in our study was

previously indicated to bind non-specifically to a small proportion of AKAP8 (Zhang et

al., 2009 Neuron 61: 385-396). This experiment reconfirms our observation that GluK2,

but not GluK1 is co-immunoprecipitated with KCC2, regardless of antibody used. (c) Co-

immunoprecipitation experiments performed in HEK-293 cells transfected with KCC2

and KAR subunits (similar to experiment in Fig. 1b), immunoprecipitated with anti-

GluK1 and GluK2, and immunoblotted with the antibodies indicated at right (KCC2,

GluK2/3, myc).

Supplemental Figure 2 (Related to Figure 2): Native KCC2 and KARs exist in the

same oligomeric complex.

(a) Native KCC2 exists as monomers, dimers, trimers and tetramers in addition to higher

order macromolecular complexes. Higher exposure of Fig. 2a, including a third native

detergent dodecyl maltoside (DDM), indicating the existence of multiple higher order

macromolecular assemblies (above ~400kDa) in vivo. (b) Antibody-shift assay

demonstrating that antibodies for the transferrin receptor did not shift GluK2/3. (c)

Antibody-shift assay similar to (Figure 2bii) using digitonin-solubilized hippocampal

membrane fractions, but with antibodies targeted to IgG or KCC2. Representative

example of three independent biological replicates; (d) Antibody-shift assay (similar to

Supp Fig. 2c) with antibodies targeted to KCC2 or IgG, but using digitonin-solubilised

cortical membrane preparations. Representative example of two independent biological

replicates.

Supplemental Figure 3 (Related to Figure 3): GluK1/2-null hippocampal neurons

have an increased monomeric:oligomeric KCC2 ratio. (ai) Higher exposure of Fig. 3a indicating the presence of a higher-order KCC2 oligomer

migrating above ~400kDa, in wild-type lane (red arrow) but undetectable in GluK1/2-/-

lane. (aii) Individual biological triplicates corresponding to Fig. 3b. Lanes used for Fig.

3b are highlighted by dotted lines. (aiii) Total KCC2 levels in RIPA solubilized

hippocampal membranes. (aiv) Schematic representation of the experimental conditions

in figures S3ai, S3aii and S3aiii. (bi) Quantitative RT-PCR of KCC2 mRNA levels

normalized to GAPDH levels, and (bii) semi-quantitative KCC2 mRNA levels from WT

and GluK1/2-/-

hippocampal lysates.

Supplemental Figure 4 (Related to Figures 3 and 4): Neto2 levels are decreased in

GluK1/2-/-

hippocampal lysates. (ai) Analysis of Neto2 protein expression in C12E9 solubilized GluK1/2

-/- hippocampal

membranes. (aii) Summary figure showing a reduction in Neto2 protein levels in

GluK1/2-/-

hippocampal lysates, after normalizing with respective tuj1 level (n=4).

Summary figure represents mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001. (b)



Example of analysis performed to determine the location of the puncta relative to the

surface (as determined by the edge of the GFP). Vectors are emanating from the centroid

and passing through individual puncta.

Supplemental Figure 5 (Related to Figure 5): Neuronal activity patterns are the

same in wild-type and GluK1/2 KO neurons. (ai) Sample image of the analysis window used to quantify neuronal activity. When the

Fluo4-AM intensity increased over 2000 arbitrary units during the acquisition period the

neuron was scored as active. (aii) Summary of all analysis similar to (ai) demonstrating

the % of neurons active in two different strains of wild-type neurons (129SVE and

C57/Bl6) and GluK1/2KO neurons. Analysis was performed on: WT 129SVE n = 115;

GluK1/2KO n = 64; WT C57/Bl6 n = 123. N values are a combination of three separate

experimental repetitions. (bi) Efficiency of knockdown of exogenous GluK2 using

scrambled shRNA and mouse-GluK2 shRNA #3 in HEK293 cells. Histogram represents

densitometric analysis of GluK2 levels upon shRNA co-transfection, normalized to

GluK2 transfections alone (n=4). (bi) GluK2 shRNA#3 does not non-specifically silence

exogenous expression of GluK1, KCC2 and KCC4 in HEK293 cells (n=3). GluK2-

shRNA#3 was subsequently used for electrophysiological experiments in Fig 5b.

Summary figure represents mean ± sem. *P < 0.05, **P < 0.01, ***P < 0.001

8



9

SUPPLEMENTAL EXPERIMENTAL PROCEDURES

Animals & approvals: All experiments were performed in accordance with approval and

guidelines from the University of Toronto Animal Care Committee and the Canadian

Council on Animal Care. Animals of both sexes were used to prepare hippocampal

cultures; all other experiments were performed on male mice. The following animal

species were used:

Wild-type C57/Bl6 (Charles River; Wilmington, MA)

Wild-type 129/SV (Charles River; Wilmington, MA)

GluK1/2-/-

maintained on a mixed 129SV/C57Bl6 background (obtained from

Chris McBain, NIH)

KCC2+/-

maintained on a mixed 129SV/C57Bl6 background (provided by Eric,

Delpire, Vanderbilt Univ)

Antibodies: See Supplemental Table 1 for complete details for all antibodies used in this

study.

RNA extraction, cDNA synthesis, and analysis of KCC2 expression by real-time PCR:

RNA was isolated from hippocampi of ~30 -old male wild-type and GluK1/2−/−

mice

using TRIzol (Invitrogen Life Technologies) according to the manufacturer’s

instructions. cDNA was synthesized using Omniscript reverse transcriptase (Qiagen)

according to the manufacturer’s instructions, using 2 μg of total RNA. 30, 60, 120ng of

cDNA samples were use in quantitative real-time PCR. cDNA samples were amplified in

triplicate using SYBR Green PCR Master Mix (Applied Biosystems) and detected with

ABI/PRISM 7900 HT cycler (Applied Biosystems). Primer Express, version 2.0,

software (Applied Biosystems) was used for primer design: 5′-

GCCATTCACAGCTTCCCCAAACTTG-3′ (KCC2 reverse), 5′-

CCACCTCTGCTGTCTACATCAGCTC- 3′ (KCC2 forward), 5′-

GCAAAGTGGAGATTGTTGCCAT- 3′ [glyceraldehyde-3-phosphate dehydrogenase

(GAPDH) forward], 5′-CCTTGACTGTGCCGTTGAATTT-3′ (GAPDH reverse).

Experiment in Fig. S3 are representative results from 3 independent sample preparations

from each genotype.

Native-Co-IP analysis: All biochemical preparations and centrifugations were performed

at 4 °C. Native-membrane fractions were prepared similarly to previously described

(Ivakine et al., 2013). Mouse brains (P30, male) were homogenized on ice in PBS using a

glass-Teflon homogenizer, followed by a brief low-speed centrifugation. Soft pellets

were resuspended in ice-cold lysis buffer [Tris·HCl, 50 mM, pH 7.4; EDTA, 1 mM;

protease and phosphatase inhibitor mixture (Roche)], homogenized, and centrifuged for

30 minutes at 25,000 × g. The membrane pellets were resuspended in solubilization

buffer (4Xw/v) [Tris·HCl, 50 mM, pH 7.4; NaCl, 150 mM; EDTA, 0.05 mM; 1.5%

C12E9, and protease and phosphatase inhibitor mixture], solubilized for 3 hours on a

rotating platform at 4 °C, and centrifuged for 1 hour at 25,000 × g. For KCC2-

coimmunoprecipitation, membrane fractions (∼0.5 –1.5 mg protein) were incubated with

anti-KCC2 (clone N1/12) (4 hours at 4 °C) with rotation, and subsequently incubated

with 20μl GammaBind IgG beads (1 hour at 4 °C). Following antibody binding to beads,



10

the beads were washed once with solubilization buffer containing 1.5% C12E9, once with

solubilization buffer minus C12E9. Bound proteins (IP fraction) were eluted with SDS

sample buffer containing DTT (1 hour min at 37 °C) and they were separated along with

1% of input fraction and 1% of unbound fraction in a standard 8% SDS-PAGE, followed

by Western immunoblotting. Experiments in Fig. 1a are representative results from 3

independent biological replicates.

HEK-293-Co-IP analysis: For co-IP experiments, HEK-293 cells were transfected with

KCC2, GluK2, GluK1b-myc (0.25 μg each) using Lipofectamine (Invitrogen) at 70%

confluency. Thirty-six hours after transfection, cells were washed with ice-cold 1× PBS

and lysed in modified RIPA buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM

EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% DOC, and protease inhibitors (Roche)].

Lysed cells were incubated on ice for 30 min and were centrifuged at 15,000 × g for 15

min at 4 °C. Cell lysates or solubilized membrane fractions (∼0.2 – 0.5 mg protein) were

incubated with Rb GluK1 and GluK2/3 antibodies (or anti-KCC2) on a rotating platform

(4 h, 4 °C). Lysates were subsequently incubated with 20μl GammaBind IgG beads (GE

Healthcare) on a rotating platform (1 h at 4 °C). After incubation, beads were washed

twice with modified RIPA buffer, and twice with modified RIPA buffer minus

detergents,. Bound proteins were eluted with SDS sample buffer and subjected to

SDS/PAGE along with 10% of input fraction and immunoblotted. Experiments in Fig.

1b, S1c are representative results from 4 and 3 independent biological replicates

respectively.

PFO-PAGE for resolving monomeric:oligomeric KCC2 ratio: All biochemical

preparations, centrifugations and PFO-PAGE were performed at 4 °C as described

(Uvarov et al., 2009) with modifications. Briefly, the membrane fractions from the

hippocampi of P30 – 60, male, WT and GluK1/2-/-

mice samples were prepared as above

using 1.5% C12E9- based solubilization buffer supplemented with 25mM iodoacetamide

to prevent the formation of artificial di-sulphide bonds. The 2x sample buffer contained

8% (w/v) sodium perfluoro-octanoate (NaPFO, Fluorochem Ltd., Derbyshire, UK),

0.004% bromphenol blue, 20% (v/v) glycerol, and 100 mM Tris base. Proteins were

separated using 5% and 7% Criterion Tris-HCl gels (Bio-Rad) in ice-cold PFO-glycine

buffer (25 mM Tris, 192 mM glycine, 0.5% NaPFO, pH 8.5), followed by Western

immunoblotting. Experiments in Fig. 3a, b, S3b are representative results from 3

independent biological replicates.

Antibody-shift assay & BN-PAGE analysis: All biochemical preparations,

centrifugations and 1D-BN-PAGE were performed at 4 °C. Native-membrane fractions

were prepared similarly as described (Swamy et al., 2006) with minor modifications.

Briefly, mouse brain / cortex / hippocampus (P30) were homogenized on ice in PBS

using a glass-Teflon homogenizer, followed by a brief low-speed centrifugation. Soft

pellets were resuspended in ice-cold lysis buffer [Bis-tris, 20mM, pH7; EDTA, 0.2mM;

sucrose, 300mM; protease and phosphatase inhibitor mixture (Roche)], homogenized,

and centrifuged for 30 minutes at 25,000 × g. The membrane pellets were resuspended in

solubilization buffer (4x w/v) [Bis-tris, 20mM, pH7; 6-aminocaproic acid, 500mM; NaCl,

20mM; EDTA, 0.2mM; glycerol, 10%; iodoacetamide, 25mM; 1% dodecyl-maltoside or



1% digitonin or 1.5% C12E9; protease and phosphatase inhibitor mixture], solubilized for

3 hours on a rotating platform at 4 °C, and centrifuged for 1 hour at 25,000 × g. Ten to

twenty µg of proteins and native MARK unstained protein standard (Invitrogen) were

mixed with BN-PAGE sample buffer (bis-Tris, 200mM; 6-aminocaproic acid, 1M;

Coomassie blue G250, 5%; sucrose, 30%; iodoacetamide, 25mM) and loaded on a linear

3-15% precast native bis-Tris gel (Invitrogen). Samples were separated in a 1D-BN-

PAGE as described (Swamy et al., 2006), using separate cathode buffer (bis-Tris, 15 mM,

pH7; Tricine, 50 mM; Coomassie blue G250, 0.02%), and anode buffer (bis-Tris 50mM,

pH7). Experiments in Fig. 2a are representative results from 3 independent biological

replicates.

Antibody-shift assay and 2D BN-PAGE analysis were performed as described (Schwenk

et al., 2012; Schwenk et al., 2009) with minor modifications. Briefly, 50μg - 100μg of

digitonin or C12E9 solubilized complexes were pre-incubated for 1 hour with 10μg of Ms-

anti-KCC2(N1/22) / Ms IgG whole molecule; and 5μg Rb-anti-GluK2/3(NL9) / Rb IgG

whole molecule / Rb-anti-HA respectively, prior to the addition of Coomassie blue G250.

1D-BN-PAGE were performed as described above using home-made 4% and 5% bis-tris

gels as described (Swamy et al., 2006). After the completion of the gel run, excised BN-

PAGE lanes were equilibrated in Laemmli buffer containing SDS and DTT for 15

minutes at room temperature to denature the native proteins. After a brief rinse in SDS-

PAGE running buffer, the excised BN-PAGE lanes were placed on a 6% or 8% SDS-

PAGE gel for separation in the second dimension. After standard electroblotting of SDS-

PAGE-resolved samples on nitrocellulose membrane, the blot was cut into two molecular

weight ranges; the top blots were subjected to western blotting analysis with Rb anti-

KCC2, Rb anti-GluK2/3 and the bottom blots with Rb anti-Neto2. Antibody-shift

experiments using hippocampal membranes are representative results from 3 independent

biological replicates, experiments using cortical membranes are representative results

from 2 independent biological replicates.

Surface biotinylation assay for studying surface: internal KCC2 ratio: Biotinylation

studies were performed as previously described (Ng et al., 2009) with modifications.

Briefly, 300μm coronal slices from age-matched (postnatal day 30 – 40) male wild-type

and GluK1/2-null littermate mice (n=5) were cut using a Leica Vibratome in modified

ACSF (180 mM sucrose, 25 mM sodium bicarbonate, 25 mM glucose, 2.5 mM KCl, 1.25

mM sodium phosphate, 2 mM MgCl2, 1 mM CaCl2, 0.4 mM sodium ascorbate, and 3

mM sodium pyruvate, and saturated with 95% O2/5% CO2 (pH 7.4. osmolarity

~295mOsm). Twelve slices from each genotype were recovered in a 50:50 mix of

modified ACSF and normal ACSF (125 mM NaCl, 25 mM sodium bicarbonate, 25 mM

glucose, 2.5 mM KCl, 1.25 mM sodium phosphate, 1 mM MgCl2, 2 mM CaCl2 (pH 7.4.

osmolarity ~295mOsm and saturated with 95% O2/5% CO2) for 30min, followed by a

second recovery in normal ACSF for another 30 min at room temperature.

Slices were incubated in 5 ml of cold-ACSF containing 500 μg/ml EZ-Link Sulfo-NHS-

SS-Biotin (21328, Thermo Scientific) bubbled in 95% O2 5% CO2, with gentle agitation

for 2 hrs at 4◦C. The reaction was stopped by quenching excess biotin in cold-ACSF

containing 100mM Tris and slices were washed twice in cold-ACSF, once in cold-

11



modified ACSF and snap-frozen immediately on dry ice. Entire cortex region was

dissected under dissecting microscope (Olympus), lysed immediately in modified RIPA

buffer [50 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1%

SDS, 0.5% DOC, protease inhibitors and phosphatase inhibitor mixture (Roche)] and

incubated on ice for 30 min. After thoroughly homogenizing, the samples was

centrifuged, supernatant was collected, and quantified using the BioRad protein

quantification kit. 50 μg of total protein in a total volume of 300 μl (made up using

modified RIPA) was mixed with 200 μl of 50% slurry of Neutravidin beads (29201,

Thermo Scientific) and rotated for 2 hrs at 4◦C. The beads were harvested by

centrifugation and the supernatant corresponding to ~5 μg of unbound fraction was

aliquoted (to measure the internal, unbiotinylated fraction of surface proteins). The beads

were subsequently washed three times in modified RIPA buffer. After the last wash all

solution was thoroughly removed from beads, and the biotin-bound and unbound

fractions were denatured in 6XSDS sample buffer containing DTT at 37◦C for hr before

resolving them on onto 6% SDS-PAGE. Subsequent immunoblot analysis was performed

as described earlier. In a small subset of this experiment, the slices were lysed, and the

neutravidin pull-down were performed in C12E9 based buffer, and we observed similar

results to Fig. 4a (data not shown).

Hippocampal cultures and electrophysiology: Low-density cultures of dissociated

mouse hippocampal neurons were prepared as previously described (Acton et al., 2012),

in accordance to guidelines from the University of Toronto Animal Care Committee and

the Canadian Council on Animal Care. Experiments were performed after 10-13 days in

culture. Cultured neurons were selected for electrophysiology based on three criteria.

Using DIC microscopy we selected neurons: (1) with a healthy oval or pyramidal shaped

cell body; (2) multiple clearly identifiable processes; and (3) a cell body and proximal

dendrites that were relatively isolated (e.g. not in the direct vicinity of other neurons).

Gramicidin (50μg/ml) perforated patch clamp recordings were performed using glass

pipettes as previously described (Acton et al., 2012). Cultured neurons were continuously

perfused with standard extracellular solution (XSC). Recordings started when the series

resistance dropped below 50 MΩ. IV-curves were made by depolarizing the membrane

potential in steps, while simultaneously stimulating GABAergic transmission. A 10μM

GABA puff was applied to the soma. A linear regression of the IPSC/P amplitude was

used to calculate the voltage dependence of IPSC/Ps; the intercept of this line with the

abscissa was taken as EGABA, and the slope of this line was taken as the synaptic

conductance. The maximum current amplitude was taken as the largest absolute current

recorded during the recordings performed for the EGABA measurement.

Electrophysiological values have not been corrected for the liquid junction potential of ~7

mV.

GluK2shRNA and Neuronal transfection: Several (mouse) GluK2 shRNA were

generated in pGIPZ backbone with a GFP reporter. They were screened in HEK-293 to

identify the shRNA with maximum target silencing and least non-specific silencing by

co-expressing with exogenous GluK2 or other negative control plasmids (GluK1, KCC4

(Karadsheh et al., 2004)) at a target:shRNA ratio of 1: 8. GluK2 shRNA clone#3

12



corresponding to mature antisense – AGAGATTGATCTTCTGTGT (Cat# RMM4431-

200414968, V3LMM_424318, Thermo Scientific), induced efficient target silencing (as

shown in supplemental figure 5b) when compared with scrambled shRNA (#RHS4346,

ThermoScientific). TransfectinTM Lipid Reagent (Biorad) was used to transfect GluK2

shRNA clone#3 or non-silencing shRNA into cultured hippocampal neurons, according

to the manufacturer’s instruction. Experiments in Fig. S5b are representative results from

3 - 4 independent biological replicates.

Fixed immunostaining and confocal microscopy: Fixed immunostaining was performed

by fixing DIV 12-14 cultured hippocampal neurons in 4% paraformaldehyde for 10 min

on ice followed by washing with 2X-PBS. Neurons were then permeabilized with PBS

containing 10% goat serum and 0.5% Triton X-100 for 30 minutes, followed by a 45 min

incubation with anti-GluK1/2 (Abcam) and mouse anti-KCC2 (N1/12) antibodies at 37◦C

to detect endogenous proteins. A small subset of experiments were also performed with

rabbit anti-GluK2/3 (Millipore) and mouse anti-KCC2 (N1/12) antibodies. Finally,

neurons were washed and incubated with Cy3-conjugated goat anti-rabbit and Alexa

Fluor 488-conjugated goat anti-mouse antibodies for 45 minutes at 37◦C. Neurons were

imaged on a Wave FX spinning disc confocal system (Quorum Technologies) with a

Zeiss Observer Z1 inverted microscope. Neurons were selected for imaging using the

same three criteria identified above under the subheading ‘Hippocampal Neurons and

Electrophysiology’. Images were acquired using 3D Image Analysis software (Perkin

Elmer). Images were obtained using a 43x 1.4-NA oil immersion objective.

Colocalization analysis was performed on representative z-stacks selected for analysis

using the ImageJ plugin JACoP (http://rsb.info.nih.gov/ij/plugins/track/jacop.html) (Bolte

and Cordelieres, 2006). Imaging experiments were performed and analyzed in a blinded

manner. Fig 1c is a representative of confocal images obtained from 26 neurons over four

independent experiments performed using 8 coverslips obtained from 4 independent sets

of cultures (of the total number of neurons analyzed (n = 26), 19 were prepared using

anti-GluK1/2, while 5 were prepared with anti-GluK2/3.

Live immunostaining and confocal microscopy: Live immunostaining was performed as

described before (Acton et al., 2012). Briefly, cultured neurons were washed in standard

extracellular solution (XCS), prior to incubating them with mouse monoclonal anti-HA

antibody at 37 °C for 15 minutes. Following incubation with the primary antibody, cells

were again washed in XCS prior to a 15 minutes dark incubation in secondary Alexa

Fluor 555 goat anti-mouse antibody. Images were acquired after 15 minutes of incubation

in the secondary antibody. Neurons were imaged on a Wave FX spinning disc confocal

system (Quorum Technologies) with a Zeiss Observer Z1 inverted microscope using a

60x 1.4NA oil immersion objective. Images were acquired and analyzed using Volocity

3D Image Analysis software (Perkin Elmer). Z-stacks were acquired at a pixel resolution

of 1024xy with a z-step of 0.2 µm (images in Fig. 4c are from singe z-planes). Samples

were acquired sequentially using line-lambda mode at excitation wavelengths of 488 nm

and 568 nm. Anti-HA (Cy3) immunofluorescence puncta intensity was measured under

constant illumination. Puncta were identified using the object identification tool in

Volocity software. We determined the percentage of puncta proximal to the membrane by

determining the location of the puncta relative to the surface of the cell. To do this we

13

http://rsb.info.nih.gov/ij/plugins/track/jacop.html



used the analysis program to identify the centroid of the cell (based on volume). We then

drew a vector from the centroid to edge of the GFP fluorescence through the puncta. If

the puncta were at the end of the vector they were classified as being proximal to the

surface; any puncta that were part-way along the vector were considered as distal to the

surface. To be considered at the end of the vector we only selected puncta where the

vector was only visible from the intracellular side of the puncta. Imaging experiments

were performed and analyzed in a blinded manner. An example image of this analysis is

found in Fig. S4. Live immunostaining analysis was performed on: wild-type n = 23,

GluK1/2KO n = 22. Each set of n values was obtained from 5 independent sets of

cultures.

Ca2+

imaging. Hippocampal neurons were loaded with the membrane-permeable

fluorescent Ca2+

indicator Fluo4-AM (Invitrogen; Carlsbad, CA, USA) for 30 min at

37°C, 5% CO2. The Fluo4 was dissolved in dimethyl sulfoxide (DMSO) and 20%

pluronic acid to a stock concentration of 1 mM and then diluted to 1 μM in our

extracellular recording solution. Following dye-loading the cells were thoroughly washed

with extracellular recording solution. Neurons were placed into the chamber of an

inverted microscope (Olympus IX71) equipped with an Olympus 0.6 NA × 40 objective.

Fluo4 was excited at 488 nm through a monochromator (Photon Technology

International (Canada) Inc., London, ON), controlled by the ImageMaster software

(Photon Technology International (Canada) Inc., London, ON). Fluorescence emission of

labeled cells at 510 nm was detected with a 16-bit CCD camera (Cascade 650,

Photometrics, Roper Scientific, Tuscon, AZ, USA). Images of 653 × 492 pixels were

accumulated at 500–1000 ms intervals. A neuron that change by 2000 fluorescent units

over a 5 second interval was considered to be active. Imaging experiments were

performed and analyzed in a blinded manner

Statistics: Results are given as mean ± SEM. For electrophysiology data sets and

immunostaining n values represent number of neurons; a minimum of three separate

coverslips were used for each of these data sets. For Western blotting experiments are

representative of a minimum of three separate biological replicates. Statistical

significance was determined using SigmaStat software. Statistical significance was

determined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical significance for

Figs 1b, 3a, S3a, 4a, S4a, 4b, 5b, 5c was determined using the Student’s t-test (two-

tailed), while Fig 3b used the paired t-test (two-tailed), 4bii, 4bvi, S5bii used the Mann-

Whitney rank sum test.

14



SUPPLEMENTAL REFERENCES

Acton, B.A., Mahadevan, V., Mercado, A., Uvarov, P., Ding, Y., Pressey, J., Airaksinen,

M.S., Mount, D.B., and Woodin, M.A. (2012). Hyperpolarizing GABAergic transmission

requires the KCC2 C-terminal ISO domain. J Neurosci 32, 8746-8751.

Bolte, S., and Cordelieres, F.P. (2006). A guided tour into subcellular colocalization

analysis in light microscopy. J Microsc 224, 213-232.

Ivakine, E.A., Acton, B.A., Mahadevan, V., Ormond, J., Tang, M., Pressey, J.C., Huang,

M.Y., Ng, D., Delpire, E., Salter, M.W., et al. (2013). Neto2 is a KCC2 interacting

protein required for neuronal Cl- regulation in hippocampal neurons. Proceedings of the

National Academy of Sciences of the United States of America 110, 3561-3566.

Karadsheh, M.F., Byun, N., Mount, D.B., and Delpire, E. (2004). Localization of the

KCC4 potassium-chloride cotransporter in the nervous system. Neuroscience 123, 381-

391.

Ng, D., Pitcher, G.M., Szilard, R.K., Sertie, A., Kanisek, M., Clapcote, S.J., Lipina, T.,

Kalia, L.V., Joo, D., McKerlie, C., et al. (2009). Neto1 is a novel CUB-domain NMDA

receptor-interacting protein required for synaptic plasticity and learning. PLoS Biol 7,

e41.

Schwenk, J., Harmel, N., Brechet, A., Zolles, G., Berkefeld, H., Muller, C.S., Bildl, W.,

Baehrens, D., Huber, B., Kulik, A., et al. (2012). High-resolution proteomics unravel

architecture and molecular diversity of native AMPA receptor complexes. Neuron 74,

621-633.

Schwenk, J., Harmel, N., Zolles, G., Bildl, W., Kulik, A., Heimrich, B., Chisaka, O.,

Jonas, P., Schulte, U., Fakler, B., et al. (2009). Functional proteomics identify cornichon

proteins as auxiliary subunits of AMPA receptors. Science 323, 1313-1319.

Swamy, M., Siegers, G.M., Minguet, S., Wollscheid, B., and Schamel, W.W. (2006).

Blue native polyacrylamide gel electrophoresis (BN-PAGE) for the identification and

analysis of multiprotein complexes. Science's STKE : signal transduction knowledge

environment 2006, pl4.

Uvarov, P., Ludwig, A., Markkanen, M., Soni, S., Hubner, C.A., Rivera, C., and

Airaksinen, M.S. (2009). Coexpression and heteromerization of two neuronal K-Cl

cotransporter isoforms in neonatal brain. The Journal of biological chemistry 284, 13696-

13704.

15



16

SUPPLEMENTAL TABLE 1: ANTIBODY INFORMATION

Antibody information Application and corresponding figures

Antibody Product

# Supplier WB IP IF

anti-KCC2

(rabbit)

07-432 Millipore (1:1000)

Figures: 1a, 3a, 3b,

4ai, S2ci, S3a

(2μg)

Figures: 1b (1:1000)

Figures: 3d

anti-KCC2

(mouse)

clone

N1/12

UC

Davis/NIMH/Neur

oMab Facility

(1:500)

Figures: 1b, S1c, 2a,

2bii, S5bii

(5-10 μg)

Figures: 1a,

S2c, S2d

(1:1000)

Figures: 1c, Data

not shown

(similar to 3d)

anti-Neto2

(rabbit)

n/a Tang et al., 2011 (1:1000)

Figures: 1a, 2biii, 4ai,

S2d, S4a

n/a n/a

anti-GluK2/3

(rabbit)

04-921 Millipore (1:1000)

Figures: 1a, 1b, 2a,

2bi, 3a, 4ai, S2cii,

S2b, S2d

(2-5μg)

Figures:

S1c, 2bi, ii,

iii

(1:1000)

Data not shown

(similar to 1c)

anti-GluK1

(rabbit)

07-258 Millipore n/a (2μg)

Figures:

S1c

n/a

anti-GluK1/2

(rabbit)

ab-

66440

abcam (1:1000)

Figures: S1b, S5b

n/a (1:1000)

Figures: 1c

anti-myc

(mouse)

R950-25 Invitrogen (1:1000)

Figures: 1b, S1c,

S5b

n/a n/a

anti-HA

(mouse)

12CA5 Roche n/a n/a (1:100)

Figures: 4b

anti-HA

(rabbit)

PRB-

101P

Covance n/a (5μg)

Figures: 2b

n/a

anti-NKCC1

(mouse)

CloneT4 Developmental

studies hybridoma

bank

(1:1000)

Figures: 3a, S3a

n/a n/a

anti-ATP1a3

(mouse)

NB300-

540

Novus Biologicals (1:2000)

Figures: 3a, 4ai

n/a n/a

anti-Tuj1

(mouse)

MMS-

435P

Covance (1:2000)

Figures: 3a, S3a,

S4a

n/a n/a

anti-

Transferrin

(mouse)

13-6800 Invitrogen n/a (5μg)

Figures:

S2b

n/a

anti-beta actin

(rabbit)

4967 Cell Signaling (1:2000)

Figures: 4ai, S5b

n/a n/a

anti-KCC4

(rabbit)

n/a Karadsheh et al.,

2004 Neuroscience (1:1000)

Figures: S5b

n/a n/a

WB, Western blot; IP, Immunoprecipitation; IF, Immunofluorescence

http://www.sciencedirect.com/science/article/pii/S0306452203007693



17

SUPPLEMENTAL TABLE 2: Synaptic Current Amplitudes and Conductance

Genetic Deletion Acute Knockdown

WT GluK1/2 KO P Scrambled

shRNA

GluK2

shRNA P

GABAergic

Synaptic

Conductance

(pS)

2.53 ± 0.42 2.13 ± .34 0.5 1.65 ± .0.24 1.89 ± 0.34 0.6

Maximum

Current (pA) 65.07 ± 11.01 67.02 ± 19.0 0.6 76.77 ±10.51 104.9 ± 10.45 0.1

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