The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines

Edited* by Roger A. Nicoll, University of California, San Francisco, CA, and approved August 9, 2011 (received for review May 25, 2011)
August 30, 2011
108 (37) 15474-15479

Abstract

The K-Cl cotransporter KCC2 plays an essential role in neuronal chloride homeostasis, and thereby influences the efficacy and polarity of GABA signaling. Although KCC2 is expressed throughout the somatodendritic membrane, it is remarkably enriched in dendritic spines, which host most glutamatergic synapses in cortical neurons. KCC2 has been shown to influence spine morphogenesis and functional maturation in developing neurons, but its function in mature dendritic spines remains unknown. Here, we report that suppressing KCC2 expression decreases the efficacy of excitatory synapses in mature hippocampal neurons. This effect correlates with a reduced postsynaptic aggregation of GluR1-containing AMPA receptors and is mimicked by a dominant negative mutant of KCC2 interaction with cytoskeleton but not by pharmacological suppression of KCC2 function. Single-particle tracking experiments reveal that suppressing KCC2 increases lateral diffusion of the mobile fraction of AMPA receptor subunit GluR1 in spines but not in adjacent dendritic shafts. Increased diffusion was also observed for transmembrane but not membrane-anchored recombinant neuronal cell adhesion molecules. We suggest that KCC2, likely through interactions with the actin cytoskeleton, hinders transmembrane protein diffusion, and thereby contributes to their confinement within dendritic spines.
The neuronal K-Cl cotransporter KCC2 transports chloride using the electrochemical gradient of K+ ions (1). In mature neurons, this action maintains a low intraneuronal chloride concentration that ensures a hyperpolarizing effect of GABA at chloride-permeable GABAA receptors. KCC2 expression, activity, and membrane traffic are tightly regulated by neuronal activity, particularly through the phosphorylation of its carboxyl-terminal domain (CTD) (24). Activation of postsynaptic glutamate receptors, for instance, reduces KCC2 activity through dephosphorylation and endocytosis within minutes (3, 5). KCC2 expression is also suppressed in pathological conditions associated with enhanced neuronal activity (6), leading to a rise in intraneuronal chloride and an alteration of GABA function (79). KCC2 therefore appears to mediate a functional cross-talk between synaptic excitation and inhibition in neurons.
Although KCC2 function primarily influences the efficacy of GABAergic signaling, its presence in dendritic spines (10) raises the question of its role in spine morphogenesis and function. Genetic ablation of KCC2 in mice compromises spine maturation and excitatory synapse formation in immature hippocampal neurons (11). This effect appears to be independent of KCC2 function but, instead, involves KCC2 interaction with the neuronal FERM-domain protein 4.1N (12). However, KCC2 expression is up-regulated during postnatal development and is maximal in mature neurons (13), after spine formation, where its role in the maintenance and function of dendritic spines remains unknown. Here, we show that suppression of KCC2 after spine morphogenesis reduces postsynaptic glutamate receptor content and relieves a constraint to the lateral diffusion and aggregation of these receptors and other transmembrane proteins within dendritic spines. This effect likely involves KCC2 interaction with submembrane actin cytoskeleton through its CTD but not its ion transport function. Thus, suppression of KCC2 in mature neurons influences synaptic efficacy at glutamatergic synapses independent of GABAergic function.

Results

Suppression of KCC2 Expression in Mature Hippocampal Neurons.

KCC2 is expressed throughout the somatodendritic membrane of cortical neurons but also in dendritic spines (10). In mature [>28 days in vitro (DIV)] hippocampal neurons, anti-KCC2 immunostaining revealed numerous KCC2-immunopositive clusters within both spine heads and spine necks (Fig. 1 A and E). The intensity of KCC2 cluster immunostaining was 76% higher in dendritic spines than on dendritic shafts (P < 0.001), suggesting that KCC2 primarily aggregates in dendritic spines. We evaluated the role of KCC2 in dendritic spine maintenance and function using RNAi. In cultured hippocampal neurons, excitatory synapses are formed from 5 to 7 DIV, whereas mature synapses onto dendritic spines appear from 8 to 10 DIV (14). Neurons were thus transfected at 14 DIV with plasmids expressing GFP and either nontarget shRNA or shRNA against KCC2 and were processed 10 d later. The efficacy and specificity of RNAi on KCC2 expression were established at the protein level (Fig. 1 CE) by comparing KCC2 and MAP2 immunoreactivity in neurons expressing either construct. We selected one of four shRNA sequences leading to maximal reduction of KCC2 immunoreactivity (−86.4 ± 4.1% of control; P < 0.005). Overexpression of this sequence had no significant effect on MAP2 immunoreactivity (P = 0.2).
Fig. 1.
Suppression of KCC2 expression in mature hippocampal neurons. (A) Confocal section of a 28-DIV hippocampal neuron immunostained for KCC2. (Left) asterisk shows the soma. (Right) Enlarged boxed region shows intense immunoreactivity in spines (arrowheads). (Scale bar: Left, 5 μm; Right, 2 μm.) (B) Normalized fluorescence intensity per cluster of KCC2 staining in dendritic shaft vs. spines in 30 cells from three independent cultures (***P < 0.001). (C) Effect of KCC2 silencing on protein expression in neurons at 24 DIV. Arrows and stars show dendrites and somata of transfected neurons, respectively. (Scale bar: 5 μm.) (D) Normalized fluorescence intensity per pixel of KCC2 staining in neurons expressing shNT (n = 10) or shKCC2 (n = 7) (P < 0.005). shKCC2, shRNA against KCC2; shNT, nontarget shRNA. (E) (Left) Confocal images of dendritic spines in neurons expressing shNT or shKCC2 showing GFP (green) and KCC2 (red) immunostaining. (Right) Line scans (from white dotted lines) of KCC2 immunofluorescence. The vertical axis shows raw KCC2 fluorescence intensity. (Scale bar: 0.5 μm.) (F) 3D reconstructions from tertiary dendrites of neurons expressing shNT or shKCC2 and GFP. (Scale bar: 5 μm.) (G) Quantification of spine length and head diameter from neurons expressing shNT or shKCC2.
KCC2 suppression in mature neurons had no significant effect on the mean density (P = 0.1) or length (P = 0.2) of dendritic spines but caused a 30% increase in spine head diameter (P < 0.001) and an increase in the proportion of mushroom-type spines (Fig. 1 F and G and Table S1). This effect contrasts with the genetic ablation of KCC2, which prevents dendritic spine morphogenesis in immature neurons, leading to predominant long filopodia-like protrusions (11). We asked whether this discrepancy was related to the mode or the timing of KCC2 suppression. In hippocampal neurons transfected at 4 DIV, before spine formation, suppression of KCC2 resulted in a 12% increase in spine length (P < 0.005) and a modest but significant reduction in spine head diameter (P < 0.05; Fig. S1). The overall spine density was unchanged (P = 0.9), but the proportion of filopodia-like protrusions was increased in neurons expressing shRNA against KCC2 (P < 0.02; Table S1). Therefore, suppression of KCC2 expression has opposite effects on spine morphology in mature vs. immature hippocampal neurons and KCC2 is not required for the anatomical maintenance of mature dendritic spines.

Reduced Quantal Size and GluR1 Accumulation in Dendritic Spines After KCC2 Suppression.

Dendritic spine morphology has been correlated with synaptic function, particularly with postsynaptic density (PSD) size (15), postsynaptic receptor content (16), and lateral diffusion (17). Therefore, increased spine head volume upon KCC2 suppression might be expected to correlate with an increased number of postsynaptic receptors and synaptic strength (18). We tested this hypothesis by recording miniature excitatory postsynaptic currents (mEPSCs) from hippocampal neurons expressing nontarget or KCC2-specific shRNAs (Fig. 2 A and B). Surprisingly, mEPSC amplitude was not increased but rather reduced in neurons expressing shRNA against KCC2 compared with nontarget shRNA (12.4 ± 0.9 pA vs. 14.9 ± 0.9 pA; P < 0.05). Suppression of KCC2, on the other hand, had no significant effect on mEPSC frequency (14.8 ± 1.9 Hz vs. 18.3 ± 2.3 Hz; P = 0.2), suggesting that the mean number of functional synapses was unaffected (Fig. 2B).
Fig. 2.
Decreased quantal size and GluR1 postsynaptic clustering upon suppression of KCC2. (A) (Left) Ten-second recordings of mEPSCs in neurons expressing nontarget shRNA or shRNA against KCC2. (Right) Scaled averages of 150 mEPSCs from the same recordings revealed no change in onset or decay kinetics. (B) Summary graphs of mEPSC amplitude distributions (Left), mean amplitude, and frequency (Right) in neurons expressing shNT (n = 27) or shKCC2 (n = 23) from five experiments. Suppression of KCC2 expression reduced the amplitude of mEPSCs by ∼17% (P < 0.001 on distributions and P < 0.05 on means) without affecting their frequency (P = 0.2). (C) Representative sections of dendrites with GFP and GluR1 immunostaining. Arrowheads indicate dendritic spines. KCC2 suppression induces a reduction in GluR1 clusters in dendritic spines. (Scale bar: 1 μm.) (D) (Upper) Normalized fluorescence intensity per cluster of GluR1 staining (shNT, n = 55; shKCC2, n = 44) from three experiments (***P < 0.005). (Lower) Quantification of the proportion of spines bearing GluR1-immunopositive clusters from the same samples (P = 0.2).
Spine enlargement may increase PSD size, and thus reduce the probability of postsynaptic receptor activation by released glutamate (19). However, we detected no difference in the onset kinetics of mEPSCs in neurons expressing KCC2-specific vs. nontarget shRNA (0.97 ± 0.03 ms vs. 0.95 ± 0.03 ms; P = 0.8; Fig. 2A). We thus asked whether suppressing KCC2 affected postsynaptic receptor density, by comparing GluR1 expression in neurons expressing shRNA against KCC2 compared with nontarget shRNA (Fig. 2 C and D). GluR1 immunostaining showed marked punctae primarily on dendritic spines and, to some extent, on dendritic shafts (Fig. 2C). The relative intensity of GluR1 clusters within dendritic spines was significantly reduced in neurons expressing KCC2-specific shRNA (−46.9 ± 8.7%; P < 0.005; Fig. 2D). In contrast, the proportion of dendritic spines bearing GluR1 clusters was unaffected (83.1 ± 3.2% vs. 88.4 ± 2.1%, respectively; P = 0.2), consistent with the lack of effect of KCC2 suppression on mEPSC frequency. These results suggest that the reduction in EPSC quantal size induced by KCC2 suppression reflects a reduced density of postsynaptic AMPA receptors at excitatory synapses.

Preventing Molecular Interactions of KCC2 with Intracellular Partners Mimics KCC2 Suppression.

KCC2 suppression may increase chloride concentration (20) and thereby reduce GABAergic signaling. This may subsequently lead to scaling of excitatory synapses through homeostatic plasticity (21). We tested this hypothesis using chronic application of the KCC2-specific antagonist VU0240551 (22). Treatment of 21 DIV neurons with 6 μM VU0240551 in DMSO for longer than 72 h induced a significant increase in spine head volume compared with treatment with DMSO alone (P < 0.001; Table S1). However, no significant change was observed in the mean amplitude (18.7 ± 1.1 pA vs. 16.8 ± 0.8 pA; P = 0.2), frequency (30.1 ± 3.1 Hz vs. 31.3 ± 3.0 Hz; P = 1.0), or onset kinetics (1.03 ± 0.02 ms vs. 1.07 ± 0.02 ms; P = 0.2) of mEPSCs (Fig. 3 A and B). Consistent with these observations, VU0240551 did not induce detectable changes in GluR1 cluster immunofluorescence in dendritic spines (+7.3 ± 8.9% of control; P = 0.2) or in the proportion of GluR1-immunopositive spines (84.1 ± 1.0% vs. 82.6 ± 1.2%; P = 0.7; Fig. 3 C and D). Therefore, decreased excitatory synapse efficacy upon suppression of KCC2 expression does not result from the loss of KCC2 function and subsequent reduction of GABA signaling.
Fig. 3.
Effect of KCC2 suppression is independent of KCC2 function. (A) Scaled averages of 100 mEPSCs from neurons treated for >72 h with either 6 μM VU0240551 in DMSO or DMSO alone. (B) Summary graphs of mEPSC amplitude (Left) and frequency (Right) in neurons treated with DMSO (n = 23) or VU0240551 (n = 24) from four experiments. KCC2 antagonist did not significantly affect mEPSC amplitude (P = 0.2) or frequency (P = 0.8). (C) GFP and GluR1 immunostaining of hippocampal neurons treated with either DMSO or VU0240551. Arrowheads indicate dendritic spines. (Scale bar: 1 μm.) (D) (Upper) Normalized fluorescence intensity per cluster of GluR1 staining in neurons treated with either DMSO (n = 44 cells) or VU0240551 (n = 40 cells) from three experiments (P = 0.2). (Lower) Quantification of the proportion of spines bearing GluR1-immunopositive clusters from the same samples (P = 0.7).
The C-terminal domain of KCC2 interacts with the 4.1N protein (11), a neuronal FERM-domain protein (12) that binds both actin and the GluR1 subunit of AMPA receptors (23). Because stabilization of synaptic AMPA receptors depends critically on the actin cytoskeleton (24), direct or indirect interaction between AMPA receptors, actin, and KCC2 may influence the maintenance of synaptic AMPA receptors. We therefore aimed at preventing KCC2-4.1N interaction without affecting KCC2 function by overexpressing the KCC2-CTD (11). We first verified that overexpressing KCC2-CTD did not affect KCC2 function by comparing the gradient in reversal potential of GABA currents (EGABA) induced at the soma vs. distal dendrites using local photolysis of caged GABA (11). In our conditions, the EGABA evoked by photolysis of RuBi-GABA (Ascent Scientific) onto the soma of hippocampal neurons was −51.3 ± 1.6 mV (n = 25; Fig. 4A), close to the equilibrium potential for Cl (−52.3 mV). In neurons expressing GFP only, we measured a gradient of 7.3 ± 1.6 mV per 100 μM between somatic and dendritic EGABA (Fig. 4 A and B). This gradient was not significantly different in neurons overexpressing KCC2-CTD (7.2 ± 2.3 mV per 100 μM; P = 0.6). In contrast, it was strongly reduced in neurons expressing shRNA against KCC2 vs. nontarget shRNA (1.7 ± 1.1 mV vs. 6.6 ± 1.5 mV per 100 μM; P < 0.05) and in cells exposed to the KCC2 antagonist VU0240551 vs. DMSO alone (2.5 ± 0.5 mV vs. 6.4 ± 0.7 mV per 100 μM; P < 0.005). Therefore, overexpressing KCC2-CTD in hippocampal neurons does not affect their apparent chloride extrusion capacity, suggesting that KCC2 transport is functional.
Fig. 4.
Overexpression of KCC2-CTD mimics the effects of KCC2 suppression. (A) (Left) Currents evoked by local photolysis of RuBi-GABA on the soma or distal dendrite of somatically whole-cell patch-clamped neurons. (Right) Representative currents at voltage steps ranging from −95 to −35 mV, with corresponding normalized current/voltage relations. Note the leftward shift in current/voltage relations of dendritic vs. somatic GABA currents. (B) Summary data showing a similar shift in neurons expressing GFP vs. KCC2-CTD (P = 0.6) but not in neurons expressing KCC2-specific vs. nontarget shRNA (*P < 0.05) or in neurons treated with VU0240551 (6 μM) vs. DMSO alone (**P < 0.005). n = 10–12 cells for each condition. (C) Scaled averages of 100 mEPSCs recorded from neurons expressing either GFP or KCC2-CTD and GFP. (D) Mean mEPSC amplitude (Left) and frequency (Right) charts. Overexpression of KCC2-CTD significantly reduced mEPSC amplitude by ∼19% (*P < 0.05) but not frequency (P = 0.2). n = 22 KCC2-CTD cells and 20 GFP cells. (E) Representative sections of dendrites with GFP and GluR1 immunostaining. Arrowheads show dendritic spines. (Scale bar: 1 μm.) (F) Normalized fluorescence intensity per cluster of GluR1 staining (Upper, **P < 0.005) and proportion of GluR1-immunopositive spines (Lower, P = 0.4) in neurons expressing GFP (n = 28) or KCC2-CTD and GFP (n = 30) in two independent experiments.
Overexpression of KCC2-CTD did not cause any significant change in the morphology of dendritic spines (Table S1) but decreased mEPSC amplitude to a similar extent as did suppression of KCC2 expression by RNAi (10.6 ± 0.3 pA vs. 13.1 ± 0.9 pA; P < 0.05; Fig. 4 C and D). The effect of KCC2-CTD was not associated with significant changes in mEPSC frequency (15.8 ± 1.6 Hz vs. 14.3 ± 3.0 Hz; P = 0.2) or rise time (0.88 ± 0.04 ms vs. 0.93 ± 0.0.3 ms; P = 0.1). Accordingly, GluR1 immunostaining showed reduced cluster intensity in the dendritic spines of neurons expressing KCC2-CTD vs. GFP only (−22.3 ± 8.1%; P < 0.005), whereas the proportion of GluR1-immunopositive spines was unaffected (90.1 ± 1.6% vs. 85.7 ± 3.1%; P = 0.4; Fig. 4 E and F). Therefore, suppressing KCC2 expression or interfering with KCC2 interaction with cytoplasmic partners without affecting its transport function reduces the strength of excitatory synapses. This effect likely reflects reduced aggregation of GluR1-containing AMPA receptors in dendritic spines.

Increased Lateral Diffusion of GluR1 Subunit in Dendritic Spines upon KCC2 Suppression.

Synaptic AMPA receptor content is influenced by interactions with anchoring proteins, exocytosis, endocytosis, and lateral receptor diffusion in the plasma membrane (25, 26). KCC2 might then contribute to diffusional constraints for AMPA receptors in dendritic spines by interacting with 4.1N and actin cytoskeleton (17). We tested this hypothesis by monitoring AMPA receptor lateral diffusion with quantum dot (QD)-based single-particle tracking (27). Neurons were cotransfected with vectors expressing recombinant GluR1 GFP-tagged at its extracellular N terminus and either nontarget or KCC2-specific shRNA constructs. QD-bound recombinant GluR1 displayed heterogeneous diffusion behaviors in dendritic spines and shafts, as do native AMPA receptors (28). In dendritic spines, some receptors located in spine heads diffused slowly, exploring only a restricted membrane area (e.g., gray trajectory in Fig. 5B). Others, although confined to the spine head, diffused more rapidly over a larger membrane area (gray trajectory in Fig. 5C). These distinct behaviors may reflect differential synaptic anchoring or trapping of receptors in endocytic zones (28, 29). We therefore distinguished slowly (D ≤ 1.5 × 10−2⋅μm2⋅s−1] and rapidly (D > 1.5 × 10−2⋅μm2⋅s−1) diffusing receptors, where D represents the diffusion coefficient. Consistent with the preferential synaptic anchoring of GluR1 in dendritic spines (29), the proportion of slow QDs was larger in spines than on dendritic shafts (46.5 vs. 34.3%; n = 43 and n = 137 QDs, respectively).
Fig. 5.
KCC2 suppression increases the lateral diffusion of GluR1 in dendritic spines. (A) Image sequences (24 s) of slow (Upper rows) and rapid (Lower rows) QD-labeled GluR1s in dendritic spines of neurons expressing either shNT or shKCC2. QD images (green) are overlaid with outlined spine membrane (red). Surface exploration of QDs (green) is visualized on maximum intensity projections. (Scale bar: 1 μm.) Arrowheads show QDs in spine heads, arrows show QD in spine neck, and the crossed arrow shows QD in dendritic shaft. (B and C) (Left) Time-averaged mean square displacement (MSD) functions of slow (B) and rapid (C) GluR1 shown in A. GluR1 diffusion in neurons transfected with shNT (gray) or shKCC2 (black) is shown. (Insets) Reconstructed trajectories of slow (shNT, 479 frames; shKCC2, 633 frames) and rapid (shNT, 427 frames; shKCC2, 266 frames) QDs. (Right) Mean MSD functions from 20 (shNT, slow), 17 (shKCC2, slow), 24 (shNT, rapid), and 15 (shKCC2, rapid) spine trajectories. Diffusion coefficients (D) and confinement domains (E) for slow vs. rapid GluR1 in neurons expressing shNT (gray) or shKCC2 (black). KCC2 extinction increases the diffusion coefficient and confinement domain of rapid (*P < 0.05 for both parameters) but not slow (P = 0.6 and P = 0.1, respectively) GluR1 in spines.
KCC2 extinction did not affect the exploratory behavior or confinement of slow receptors in spines (Fig. 5 A and C). Their mean diffusion coefficient and confinement domain were not significantly different in neurons expressing KCC2-specific vs. nontarget shRNA (D = 0.82 ± 0.01 × 10−2⋅μm2⋅s−1 vs. 0.85 ± 0.07 ×10−2⋅μm2⋅s−1, P = 0.6; L = 97 ± 6 nm vs. 111 ± 7 nm, P = 0.1), where L represents the confinement domain. In contrast, rapid QD-bound GluR1 in dendritic spines exhibited a twofold increase in diffusion coefficient and a 1.7-fold increase in confinement domain in neurons expressing KCC2-specific vs. nontarget shRNA (D = 12.05 ± 2.45 × 10−2⋅μm2⋅s−1 vs. 5.99 ± 1.40 × 10−2⋅μm2⋅s−1, P < 0.05; L = 611 ± 13 nm vs. 366 ± 5 nm, respectively, P < 0.05; Fig. 5 D and E). This effect was also reflected by the steeper slope of mean square displacement functions derived from trajectories in either condition (Fig. 5C). Thus, KCC2 apparently constrains, directly or indirectly, lateral movements of rapid GluR1-containing AMPA receptors in dendritic spines. This effect seems to be independent of changes in spine head diameter, because control experiments showed that lateral diffusion of GluR1 was unrelated to spine size (Fig. S2). In contrast to its effect on GluR1 diffusion in spines, KCC2 suppression did not significantly alter diffusion on dendritic shafts. Thus, the mean diffusion coefficient and confinement domain of both slow and rapid QD-bound GluR1 were not significantly different in neurons expressing KCC2-specific vs. nontarget shRNA (Fig. S3). We conclude that KCC2 constrains GluR1 lateral diffusion specifically in dendritic spines but not on dendritic shafts. This constraint primarily affects rapidly diffusing GluR1, suggesting that distinct molecular interactions restrict diffusion of slow GluR1-containing AMPA receptors.

KCC2 Constrains Lateral Diffusion of Transmembrane but Not Membrane-Anchored Neuronal Cell Adhesion Molecule in Dendritic Spines.

KCC2 molecules are densely clustered in dendritic spines (ref. 10 and our observations) and bind actin cytoskeleton through direct 4.1N interaction (11). They might then constrain lateral diffusion of membrane proteins in dendritic spines by specific cytoskeletal interactions or by nonspecific molecular crowding. To discriminate between these possibilities, we examined the lateral diffusion of two distinct isoforms of the neuronal cell adhesion molecule (NCAM). NCAM 180 is a transmembrane isoform with a short intracellular domain that interacts with the actin cytoskeleton through β1-spectrin, whereas NCAM 120 lacks an intracellular domain and is membrane-anchored by a GPI (30) (Fig. 6A). We transfected neurons with chicken NCAM 180 or NCAM 120 together with nontarget or KCC2-specific shRNA. Consistent with their distinct membrane interactions, NCAM 180 explored a smaller area than NCAM 120 in dendritic spines (Fig. 6 B and C) and its diffusion in dendritic spines was 3.2-fold slower than that of NCAM 120 (Fig. 6D). In neurons in which KCC2 expression was suppressed by RNAi, membrane diffusion of NCAM 180 (black trajectory in Fig. 6B) but not NCAM 120 (black trajectory in Fig. 6C) was increased by almost twofold compared with neurons expressing nontarget shRNA (D = 6.62 ± 0.46 × 10−2⋅μm2⋅s−1 vs. 3.81 ± 0.32 × 10−2⋅μm2⋅s−1; P < 0.001; Fig. 6D) and their confinement domain was increased by 1.5-fold (L = 440 ± 39 nm vs. 299 ± 18 nm; P < 0.003). Because the 180 isoform is the predominant NCAM in dendritic spines (31), we also examined endogenous NCAM in spines and observed a similar increase in lateral diffusion on suppression of KCC2 (Fig. S4). In contrast, NCAM 120 diffusion was not significantly affected in these conditions (D = 15.05 ± 1.78 × 10−2⋅μm2⋅s−1 vs. 12.18 ± 1.32 × 10−2⋅μm2⋅s−1, respectively; P = 0.2; Fig. 6D). These results show that KCC2 suppression specifically facilitates lateral diffusion of transmembrane but not membrane-anchored NCAM in dendritic spines. We conclude that KCC2 constrains protein diffusion in dendritic spines, likely via interactions with submembrane proteins and cytoskeleton rather than by nonspecific molecular crowding.
Fig. 6.
KCC2 suppression increases the lateral diffusion of transmembrane but not membrane-bound NCAM in dendritic spines. (A) Schematic structure of NCAM 120 and NCAM 180. (B and C) (Upper) Representative reconstructed trajectories (Insets) and time-averaged mean square displacement (MSD) functions for recombinant NCAM 180 (B: shNT, 725 frames; shKCC2, 692 frames) and NCAM 120 (C: shNT, 248 frames; shKCC2, 307 frames) in dendritic spines of neurons expressing shNT (gray) or shKCC2 (black). (Scale bar: 1 μm.) (Lower) Mean MSD functions from 51 (shNT, NCAM 180), 66 (shKCC2, NCAM 180), 37 (shNT, NCAM 120), and 65 (shKCC2, NCAM 120) spine trajectories. (D) Diffusion coefficients (Diff. coeff.) of NCAM 180 (Left) and NCAM 120 (Right) show increased diffusion of NCAM 180 (***P < 0.001) but not NCAM 120 (P = 0.2) in neurons expressing shKCC2 (black) compared with shNT (gray). Data are from three and two independent cultures, respectively.

Discussion

Our results show that suppressing KCC2 expression in mature neurons, after spine formation, does not compromise spine maintenance but reduces the efficacy of excitatory synapses. This effect is independent of KCC2 function but reflects an alteration of AMPA receptor aggregation in dendritic spines.
In rat cortical neurons, KCC2 expression is very low at birth and increases steadily during the first 2 wk of postnatal development, inducing hyperpolarization of EGABA (13) with a timing strikingly paralleling that of spinogenesis (32). Several arguments suggest that KCC2 may differentially affect spine morphogenesis and excitatory synaptic function in neonate vs. mature neurons. Genetic ablation of KCC2 prevented spine maturation, leading to filopodia-like protrusions in immature neurons (11), whereas no evidence for compromised spine maturation was detected on chronic suppression of KCC2 after spine formation (Fig. 1), suggesting that KCC2 is required for the formation but not the maintenance of dendritic spines in hippocampal neurons. Accordingly, mEPSC frequency was reduced in KCC2−/− neurons, but suppression of KCC2 in mature neurons did not affect mEPSC frequency or the proportion of spines bearing GluR1 clusters. Therefore, the number of functional synapses seems to be unaffected by KCC2 suppression in mature neurons. However, we observed a remarkable increase in spine head volume, which was mimicked by pharmacological suppression of KCC2 function. This increase may reflect the loss of ion transport function by KCC2. K-Cl cotransport is known to be crucial for volume regulation in many cell types (33). KCCs mediate transmembrane transport of K+ and Cl ions, and thereby function as solute efflux pathways (34). Because KCC2 is the only transporter that extrudes Cl under isotonic conditions in neurons, its loss of function may then prevent osmotic regulation in dendritic spines, particularly upon cation influx through postsynaptic glutamate receptors. Although this hypothesis deserves further testing, our results predict that modulation of KCC2 function (35, 36) or trafficking (5) by synaptic activity might contribute to activity-dependent remodeling of spine volume.
Suppression of KCC2 led to reduced aggregation of postsynaptic AMPA receptors and reduced quantal size at excitatory synapses. This effect was not a consequence of the increase in spine volume because it was also observed upon overexpression of the KCC2-CTD (which did not induce increased spine volume) but not on pharmacological blockade of KCC2 function. The number and stability of synaptic AMPA receptors are controlled by several mechanisms, including (i) membrane insertion and removal through endo- and exocytosis (25), (ii) anchoring to submembrane scaffolding proteins (3739), and (iii) lateral diffusion in plasma membrane (40). Our results suggest that KCC2 interferes with GluR1 accumulation at synapses mostly through the latter mechanism. First, KCC2 suppression did not reduce lateral diffusion of GluR1 on dendritic shafts, suggesting that GluR1 was not massively trapped and endocytosed throughout the extrasynaptic membrane (29). Second, KCC2 suppression had no detectable effect on lateral diffusion of slow (D ≤ 1.5 × 10−2⋅μm2⋅s−1) recombinant GluR1 in dendritic spines. Slowly moving receptors probably form part of the synaptic receptor pool strongly anchored to subsynaptic scaffold (17, 28, 29) and, to a lesser extent, the pool of receptors immobilized in endocytic zones (29). In contrast, rapid receptors may be part of either a synaptic pool loosely interacting with the scaffold or an extrasynaptic pool located in the vicinity of the PSD and rapidly exchanging with synaptic receptors (28, 41). We suggest that by specifically constraining the lateral diffusion of rapidly moving receptors in dendritic spines, KCC2 may contribute to maintain the exchange pool of receptors within spines, and thus influence the size of the synaptically anchored pool. In fact, reduced synaptic efficacy attributable to increased AMPA receptor mobility at the spine surface was recently demonstrated at hippocampal synapses and was associated with a redistribution of AMPA receptors (AMPARs) between the PSD and perisynaptic membrane (19). In addition, a reduction in synaptic receptor content is observed following impaired filling of the recycling (extrasynaptic) receptor pool by displacement of endocytic zones from the vicinity of the PSD without affecting the diffusion of slow synaptic receptors (29).
How might KCC2 constrain lateral diffusion of AMPARs? Suppression of KCC2 expression may influence GluR1 lateral diffusion as a consequence of the morphological changes in dendritic spines. Larger spine heads may have reduced steric constraints to lateral diffusion of membrane proteins. However, this seems unlikely because spines with large heads were shown to have slower membrane kinetics than spines with small heads (42). In addition, the KCC2 antagonist VU0240551 increased spine head diameter without affecting GluR1 clustering. Finally, increased diffusion after KCC2 suppression was limited to transmembrane spine proteins (GluR1 and NCAM 180) but did not affect GPI-anchored NCAM. This observation suggests that KCC2 may specifically alter the diffusion of proteins interacting with submembrane protein partners. Several ion transport proteins have been shown to play a critical role in anchoring the actin cytoskeleton to plasma membrane in a variety of cell types. In fibroblasts, the Na+/H+ exchanger NHE1 anchors actin filaments to control cytoskeleton organization and cell shape independent of its ion transport function (43). In erythrocytes, the anion exchanger AE1 docks actin to plasma membrane through direct interaction with linker proteins of the ankyrin and 4.1 families (44). These linker proteins contain an aminoterminal FERM domain that binds transmembrane proteins, such as ion transport proteins, and a CTD that binds actin or actin-associated proteins (45). In particular, the neuronal 4.1N protein binds both GluR1 (23) and KCC2 (11). We suggest that KCC2 hinders transmembrane protein diffusion through a mechanism involving its interaction with 4.1N and actin cytoskeleton. Indeed, preventing KCC2 interactions with intracellular partners by overexpressing its CTD mimicked the effects of KCC2 suppression on GluR1 clustering and synaptic efficacy. This constraint to lateral diffusion also affects other transmembrane proteins interacting with 4.1N and/or the actin cytoskeleton, such as NCAM. KCC2 may then contribute to the membrane anchoring of a complex involving actin, 4.1N, and possibly other partners. The organization of such a complex remains to be determined, as well as the role of the direct 4.1N-GluR1 interaction, which has recently been shown to be necessary for the extrasynaptic insertion of AMPA receptors (46).
In addition to being developmentally regulated (13), KCC2 expression is suppressed in mature neurons in several pathological conditions, including epilepsy (9) and neuropathic pain (8). Suppression of KCC2 in these conditions has been suggested to induce a depolarizing shift in EGABA that might reduce the efficacy or even shift the polarity of GABAergic transmission and increase neuronal excitability (9). Our results reveal that KCC2 suppression in mature neurons may act in a homeostatic manner to reduce simultaneously the efficacy of excitatory inputs onto neurons with reduced KCC2 expression. Finally, KCC2 function has been shown to be modulated in physiological conditions both by intrinsic (47) and synaptic (36) activity. Such modulation mostly involves phosphorylation of the KCC2-CTD, which may act directly to affect ion transport function (48) or decrease KCC2 membrane stability (3, 5). Therefore, a rapid increase in PKC and Src-kinase activities associated with long-term plasticity (49, 50) may rapidly and locally modulate KCC2 function and membrane aggregation. Our results predict that this may have an impact on both spine volume through modulation of ion transport and synaptic efficacy through increased lateral diffusion of AMPA receptor in dendritic spines.

Materials and Methods

Hippocampal neurons were prepared from embryo day 19 Sprague–Dawley rat pups. Cells were plated on polyornithine-coated glass coverslips at a density of 2.5 × 104 cells⋅cm−2 and cultured at 37 °C in a CO2 incubator in Neurobasal medium supplemented with B27 (Invitrogen), 2 mM glutamine, and penicillin/streptomycin. After 4 or 14 DIV, neurons were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions (DNA/lipofectamine ratio of 1 μg/3 μL per well). Neurons were used for biological assays within 10–12 d. Additional details are available in SI Materials and Methods.

Acknowledgments

We thank J. Nabekura for rat KCC2-IRES-GFP construct; J. A. Esteban for GluR1-GFP; R. Miles, J. A. Esteban, C. Bernard, and E. Petrini for critical reading of an earlier version of the manuscript; R. M. Mège for chicken NCAM constructs and antibodies; and M. Dahan for sharing single particle tracking analysis software. This work was supported by the Institut National de la Santé et de la Recherche Médicale (Avenir program grant to J.C.P.), research grants (to J.C.P.) from the City of Paris, Federation pour la Recherche sur le Cerveau, Fyssen Foundation, and Fondation pour la Recherche Medicale. G.G. was the recipient of fellowships from the Neuropole-Ile de France and the Fondation pour la Recherche Medicale.

Supporting Information

Supporting Information (PDF)
Supporting Information

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Information & Authors

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Published in

Go to Proceedings of the National Academy of Sciences
Go to Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences
Vol. 108 | No. 37
September 13, 2011
PubMed: 21878564

Classifications

Submission history

Published online: August 30, 2011
Published in issue: September 13, 2011

Acknowledgments

We thank J. Nabekura for rat KCC2-IRES-GFP construct; J. A. Esteban for GluR1-GFP; R. Miles, J. A. Esteban, C. Bernard, and E. Petrini for critical reading of an earlier version of the manuscript; R. M. Mège for chicken NCAM constructs and antibodies; and M. Dahan for sharing single particle tracking analysis software. This work was supported by the Institut National de la Santé et de la Recherche Médicale (Avenir program grant to J.C.P.), research grants (to J.C.P.) from the City of Paris, Federation pour la Recherche sur le Cerveau, Fyssen Foundation, and Fondation pour la Recherche Medicale. G.G. was the recipient of fellowships from the Neuropole-Ile de France and the Fondation pour la Recherche Medicale.

Notes

*This Direct Submission article had a prearranged editor.

Authors

Affiliations

Grégory Gauvain
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Ingrid Chamma1
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Quentin Chevy1
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Carolina Cabezas
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Theano Irinopoulou
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Natalia Bodrug
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Michèle Carnaud
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Sabine Lévi2 [email protected]
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France
Jean Christophe Poncer2 [email protected]
Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche-S 839, F75005 Paris, France;
Université Pierre et Marie Curie, F75005 Paris, France; and
Institut du Fer à Moulin, F75005 Paris, France

Notes

2
To whom correspondence may be addressed. E-mail: [email protected] or [email protected].
Author contributions: S.L. and J.C.P. designed research; G.G., I.C., Q.C., C.C., N.B., S.L., and J.C.P. performed research; M.C. contributed new reagents/analytic tools; G.G., I.C., Q.C., T.I., S.L., and J.C.P. analyzed data; and S.L. and J.C.P. wrote the paper.
1
I.C. and Q.C. contributed equally to this work.

Competing Interests

The authors declare no conflict of interest.

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    The neuronal K-Cl cotransporter KCC2 influences postsynaptic AMPA receptor content and lateral diffusion in dendritic spines
    Proceedings of the National Academy of Sciences
    • Vol. 108
    • No. 37
    • pp. 15011-15534

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