Whether physiological patterns of neuronal activity control developmental enhancement of KCC2 function remains controversial. At least three studies have provided data supporting the idea that chronic blockade of GABAergic (Ganguly et al., 2001; Leitch et al., 2005) or glycinergic (Shibata et al., 2004) inputs in developing neuronal networks inhibits the developmental increase in KCC2 functional expression. In contrast, other studies have not found significant changes in KCC2 functional expression after prolonged inhibition of GABAergic neurotransmission (Ludwig et al., 2003; Titz et al., 2003). The later conclusion does not exclude, however, that the activity might contribute to fine adjustments of the post-translational regulation (such as phosphorylation) and activation of plasmalemmal KCC2 function. Indeed, a critical role of the brain-derived neurotrophic factor (BDNF) and other trophic factors in the control of the developmental expression of KCC2 has been shown (Kelsch et al., 2001; Ludwig et al., 2011a,b). Given that neuronal activity regulates release of BDNF from both pre- and post-synaptic terminals (Kuczewski et al., 2008, 2010; Matsuda et al., 2009), it is plausible that BDNF and/or other trophic factors contribute to activity-dependent modulation of KCC2 in a context-dependent manner.

An important process in the maturation of glutamatergic networks is the specific form of plasticity resulting from co-incident pre- and post-synaptic spiking (Debanne and Poo, 2010). Similarly, spike-time-dependent plasticity has been shown to exist for GABAergic synapses (Woodin et al., 2003). Most intriguingly, within a sub-time domain, this new form of plasticity is dependent on changes of [Cl⁻]_i. A suggested critical step in the mechanism is a rapid postsynaptic change in the efficiency of chloride extrusion by KCC2 (Fiumelli et al., 2005).

Currently there is a large number of observations describing a strong decrease in KCC2 expression in different pathologies, including temporal lobe epilepsies (Palma et al., 2006; Huberfeld et al., 2007; Bragin et al., 2009; Barmashenko et al., 2011), focal cortical dysplasia (Munakata et al., 2007; Shimizu-Okabe et al., 2011; Talos et al., 2012), ischemia (Galeffi et al., 2004; Papp et al., 2008; Jaenisch et al., 2010), as well as pathologies associated with different types of axonal injury (Coull et al., 2003; Shulga et al., 2008; Boulenguez et al., 2010). Although the primary mechanisms initiating down-regulation of KCC2 during these pathologies are not known, the common point between them is a strong decrease of mRNA and protein expression levels. The exact mechanisms triggering inactivation of the KCC2 function are not yet well understood. Several groups have shown that epileptic-like neuronal activity itself could serve as triggering event. For instance, Nardou et al. (2009) showed suppression of the KCC2 function after induction of the epileptiform-like activity in the entire immature (P5-P7) rat hippocampal preparation. Rivera et al. (2004) found out that induction of epileptic-like activity to acute hippocampal slices rapidly (30–60 min) reduced expression of KCC2 in the CA1 region of the hippocampus and suggested a critical role of the glutamate and TrkB receptors in the processes. These results, however, were updated recently by Puskarjov et al. (2012) who showed that the half–life of plasma membrane KCC2 under control conditions might be significantly longer than previously reported. The difference between the two publications may be mainly attributed to differences in slice preparation. In the work by Puskarjov and collaborators, great care was taken to minimize damage of slices during preparation. In addition, they found that the mechanism for activity-dependent degradation of plasma membrane KCC2 requires Ca²⁺-dependent activation of the protease calpain. To achieve similar levels of KCC2 down-regulation as Rivera et al. (2004), the authors had to incubate slices with a higher concentration of NMDA (100 μM) and for a longer time (4 h). At these doses, NMDA triggers several neurotoxic signaling cascades (Hardingham et al., 2002; Ivanov et al., 2006), reviewed by Medina (2007) and Hardingham and Bading (2010). A rapid NMDA receptor-dependent KCC2 inactivation was also reported in two other studies performed on cultured hippocampal neurons (Lee et al., 2011; Pellegrino et al., 2011).

Many studies reporting a global pathology-dependent down-regulation of KCC2 expression mentioned also no-change or up-regulation of KCC2 in a sub-population of neurons surviving excitotoxic or ischemic insults. For example, Conti et al. (2011) demonstrated an up-regulation of KCC2 cotransporter in human peritumoral epileptic cortex (Conti et al., 2011). Huberfeld et al. (2007) have shown robust KCC2 staining in some subicular pyramidal cells in human temporal lobe epilepsy (TLE) brain sections. Jaenisch et al. (2010) have found a high level of KCC2 expression in infarct core neurons surviving a focal ischemic insult. Papp et al. (2008) found a delayed pattern of down-regulation in interneurons in the same model. Consistent with these data, Pellegrino et al. (2011) showed that all neurons surviving excitotoxic treatment with NMDA restored their normal Cl⁻ extrusion ability and increased their expression of KCC2.

While robust pathology-like activation of a neuronal network may lead primarily to inactivation of KCC2, short activity episodes might induce functional up-regulation. For instance, Khirug et al. (2010) found that a single seizure episode in vivo, as well as brief seizure-like activity in vitro in neonates, induced a potent increase in KCC2 plasma membrane expression (with unchanged total KCC2 expression) that correlated with enhancement in the efficacy of GABAergic inhibition. The up-regulated KCC2 activity was reported also in several other studies (Khirug et al., 2005; Banke and Gegelashvili, 2008; Chorin et al., 2011; Bos et al., 2013); however, it remains unclear how the described modulations of KCC2 activity are related to physiological or pathology conditions.

Since many of the discussed above reports show that restoration of the physiological level of Cl⁻ results in normal neuronal functioning, this suggests that targeting of the KCC2 and/or NKCC1 could be an efficient therapeutic strategy (reviewed by Kahle et al., 2008; Löscher et al., 2013). Regarding this, recent studies concentrated on high-throughput screening of the compounds capable to selectively inhibit the NKCC1 (Delpire et al., 2009, 2012) and activate KCC2 (Gagnon et al., 2013). Further optimization of the reported compounds as well as creation of new agents targeting specific signaling pathways upstream of KCC2 and/or NKCC1 will make in nearest future an important breakthrough for KCC2-related therapy.

Most mature CNS neurons exhibit high levels of KCC2 expression, while low expression is detected in immature neurons (reviewed in Blaesse et al., 2009; Chamma et al., 2012). KCC2 mRNA levels are increased with neuronal maturation, KCC2 protein expression shows concordance with the mRNA level (Lu et al., 1999; Li et al., 2002; Stein et al., 2004). RNA interference approaches, such as anti-sense oligonucleotides (Rivera et al., 1999) or specific siRNAs (Bortone and Polleux, 2009; Pellegrino et al., 2011; Gauvain et al., 2011; Succol et al., 2012), lead to a reduction of KCC2 protein and a resulting loss of KCC2 activity. Thus, the control at the mRNA level is an efficient way to regulate the long-term activity of KCC2.

Selective decrease in KCC2 mRNA has been observed during neurological disorders (Palma et al., 2006; Huberfeld et al., 2007), after sustained interictal-like activity (Rivera et al., 2004), and after axonal injury (Nabekura et al., 2002; Toyoda et al., 2003; Shulga et al., 2008, 2009). Activity-dependent decreases in KCC2 mRNA might therefore be an important factor contributing to the down-regulation of KCC2 activity in certain pathological conditions. At least 10 putative transcription factor binding sites have been identified in the SLC12A5 gene encoding KCC2 promoter and proximal intron-1 regions (Uvarov et al., 2006; Uvarov, 2010). These sites are highly conserved elements among promoter regions of other mammalian KCC2 genes. Transcription factor early growth response 4 (Egr4) has been identified as a potent regulator of KCC2 expression (Uvarov et al., 2006), which mediates BDNF-dependent KCC2 transcription in immature neurons (Ludwig et al., 2011b). Another regulatory element in the KCC2 promoter is the E-box, that binds the ubiquitously expressed upstream stimulating factors USF1 and 2, thus contributing KCC2 up-regulation in the developing cortex (Markkanen et al., 2008). In addition, two neuron-restrictive silencing elements (NRSE) were found in SLC12A5; however, the data on whether they are involved in the developmental up-regulation of KCC2 are controversial (Karadsheh and Delpire, 2001; Uvarov et al., 2005; Yeo et al., 2009). The role of other putative transcription factor binding elements in the KCC2 promoter remains obscure. One important task will be to characterize in more detail the regulatory elements controlling SLC12A5 transcription.

Numerous studies suggest that, by analogy with other types of transporters and Cl⁻ channels, the CCC family members function as oligomers (McKee et al., 2000; Moore-Hoon and Turner, 2000; Casula et al., 2001; De Jong et al., 2003; Starremans et al., 2003; Simard et al., 2004; Blaesse et al., 2006; Parvin et al., 2007; Simard et al., 2007; Pedersen et al., 2008; Casula et al., 2009; Warmuth et al., 2009). With regard to KCC2, the ability of this molecule to form molecular complexes corresponding to mono-, di-, tri-, and tetrameric complexes was first demonstrated by Blaesse et al. (2006). The authors showed also a clear age-dependence of the oligomerization: the immature brains were characterized by higher content of the monomers, whereas adult tissue produced mostly oligomeric forms of the KCC2. An additional set of data demonstrating the ability of KCC2 to form dimers in vivo comes from studies of the distribution (Markkanen et al., 2013) and interaction (Uvarov et al., 2009) between KCC2a and KCC2b isoforms (Uvarov et al., 2007). Using the native perfluorooctanoate (PFO)-PAGE system, as well as a co-immunoprecipitation assay, Uvarov et al. (2009) have shown that KCC2a and KCC2b isoforms form homo- and heterodimers in vivo and in heterologous HEK-293 expression system. Despite multiple evidence describing oligomerization of KCC2, the functional context of this protein complex remains unclear. Is it critical for translocation to plasma membrane? What is the number of subunits that form the ion-transport unit? Is oligomerization involved in KCC2 surface expression and/or internalization? These and other questions remain to be answered and should be a subject for future studies.

By reviewing results of the KCC2 oligomerization studies, we have to be aware of some technical problems that one may encounter during analysis of the KCC2 using Western blot. Due to yet unidentified reasons, KCC2 is prone to forming SDS-resistant high molecular aggregates during standard protein extraction and solubilization procedures that are normally used for denaturation and dissociation of protein complexes (Gallagher, 2012). By performing Western blot analysis of the KCC2, we have found that in addition to the age-dependent changes in KCC2 oligomeric/monomeric ratio described in the above study (Blaesse et al., 2006), the ability to form oligomers strongly depends on the experimental procedure and is different for endogenously expressed KCC2 and for KCC2 overexpressed in heterologous systems (Figure 2). A very rapid protein extraction reduces the formation of the KCC2 oligomeric complexes under SDS-denaturating conditions, whereas long extraction (more than 4 min) increases the probability to get dimers. Interestingly, once the KCC2 dimer complexes are formed, we could not dissociate them to monomeric structure using boiling with SDS or in combination with high concentration (8 M) of urea at high temperature, a treatment that works efficiently to disrupt such strong molecular complexes as NMDA receptors-associated postsynaptic densities (Krapivinsky et al., 2003, 2004).

Plasma membrane insertion, surface stabilization, and internalization of the molecule constitute one more important mechanism of KCC2 regulation. Numerous studies have found that pathological-like patterns of neuronal activity initiate rapid withdrawal of the KCC2 from plasma membrane that leads to a reduction of KCC2-mediated ion-transport (Rivera et al., 2004; Lee et al., 2007, 2010, 2011; Wake et al., 2007; Puskarjov et al., 2012; Chamma et al., 2013). Using an elegant approach of visualizing KCC2 internalization harboring an extracellular tag, Zhao and colleagues have shown a critical role of the cytoplasmic C- and not N-termini for KCC2 trafficking (Zhao et al., 2008). The authors also localized in the KCC2 C-terminus a novel non-canonical di-leucine motif (₆₅₇LLXXEE₆₆₂) that seems to be essential for the constitutive internalization of the transporter and for binding to the clathrin-binding adaptor protein-2 complex. Several phosphorylation sites on the C-terminus have been implicated in mechanisms controlling KCC2 turnover (see below “phosphorylation” chapter for details and Figure 1).

The internalization of overexpressed KCC2 is controlled by the clathrin-mediated endocytosis pathway in HEK-cells (Zhao et al., 2008) and can be prevented by an inhibitor of the clathrin pathway in cultured neuronal cells (Lee et al., 2011). However, no systematic work was performed to characterize the recycling mechanism involved or the signaling pathways controlling the KCC2 membrane insertion. A recent study (Puskarjov et al., 2012) launched the discussion on the timing of the KCC2 turnover in neuronal membrane. Previous work (Rivera et al., 2004), which was performed using a biotinylation approach and hippocampal slices prepared using a slice chopper (i.e., resulting in a significant number of damaged cells), suggested that half of the KCC2 molecules are renewed in neuronal plasma membrane within 30–40 min. Using a different experimental approach and slices prepared using a vibratome (i.e., with a lower rate of neuron damage), Puskarjov et al. (2012) estimated the half time of KCC2 turnover to be much longer (several hours or even days). On the other hand, a study performed using cultured hippocampal neurons showed that 45-min treatment of neurons with dynasore, an inhibitor of the endocytosis, produced twofold increase of the surface expression of the KCC2 (Lee et al., 2010), indicating the existence of a relatively rapid constitutive KCC2 endocytosis in this model. A further evidence supporting the high rate of the KCC2 turnover was obtained in a heterologous expression system (HEK-293 cells) where over-expressed KCC2 showed a half-time turnover rate close to 10 min (Lee et al., 2007; Zhao et al., 2008). Thus, the timing and mechanisms controlling KCC2 surface expression remain to be fully elucidated.

The structural elements of KCC2's ion-transport unit are not known. Unlike other members of the KCC family (KCC1, KCC3, and KCC4), which become active only after osmotic stress (reviewed by Flatman, 2008; Blaesse et al., 2009), KCC2 is able to extrude Cl⁻ in isotonic conditions. The over-expression of this transporter in heterologous expression systems (Payne, 1997; Williams and Payne, 2004) or neuronal cells (Chudotvorova et al., 2005; Lee et al., 2005; Pellegrino et al., 2011; Acton et al., 2012) produces a clearly detectable ion flux that could be rapidly (within few seconds) and reversibly inhibited using high concentrations of furosemide, a sulfamoylbenzoic acid “loop” diuretics (Payne, 1997; Williams and Payne, 2004; Friedel et al., 2013). These data suggest that, when expressed in the plasma membrane, KCC2 operates as constitutively active K⁺-Cl⁻ transporter.

Several groups have identified in the KCC2 molecule different regions and amino acid residues that regulate the ion-transport activity. Hartmann et al. (2010) found that mutation of four evolutionarily conserved cysteine residues in the large extracellular loop of KCC2 (C287, C302, C322, C331, Figure 1) inhibited ⁸⁶Rb⁺ flux mediated by the transporter without affecting its surface expression. Interestingly, mutation of the corresponding cysteines in KCC4, which is the closest to KCC2 member of the SLC12 family, did not affect the ion transport. These data suggest a distinct organization of ion-transport mechanism in KCC2 and KCC4, thus further enhancing exclusiveness of the KCC2. An important role in KCC2-mediated ion transport plays also C568, located inside the 10th putative transmembrane domain (Reynolds et al., 2008). Mutation of this residue (C568A) makes KCC2 inactive, but it remains unclear whether C568A mutation inhibits intrinsic activity or affects membrane targeting of the molecule. A study performed by Horn et al. (2010), described a decreased interaction of the KCC2_C568A mutant with cytoskeleton-associated 4.1N protein, and suggested that the C568A mutation causes changes in the tertiary structure of the molecule. Additional analysis is necessary to clarify the mechanism of how the C568A mutation silences KCC2 activity.

As illustrated in Figure 1, the N- and C- terminal domains of KCC2 and other members of KCC subfamily, are both located intracellularly. Several sets of data suggest that both domains can regulate intrinsic activity of the transporter. Thus, by expression in Xenopus oocytes of different KCC1 constructs with truncated intracellular regions, Casula et al. (2001) found that deletion of either N- or C- termini abolished ion transport activity of KCC1 without affecting transporter expression at the oocyte surface. Consistent with this finding, Li et al. (2007) showed that truncation of the N-terminus of KCC2 inhibited its ion-transport activity and did not affect the surface expression of the protein (Figure 1). A further evidence highlighting the importance of the KCC C-terminus in controlling the intrinsic property of ion transport was found in a study by Rinehart et al. (2009). Authors showed that phosphorylation of KCC3 at two newly identified sites (T991 and T1048) strongly inhibited the ion-transport activity of the transporter without modifying its surface expression. Age-dependent changes of the phosphorylation status of equivalent threonine sites in the KCC2 molecule (T906 and T1007) were reported in the same study; however it remains unclear whether phosphorylation of the KCC2 at these residues modifies its intrinsic activity or surface expression.

The leucine 675 (L675) identified by Döding et al. (2012) is one more residue critically involved in KCC2 activity: its mutation to alanine (L675A) strongly reduced ⁸⁶Rb⁺ flux mediated by the KCC2 without affecting the surface expression of the transporter in mammalian cell lines.

It had been known for a long time that KCC2 could be constitutively active in isotonic conditions in HEK293 cells (Payne, 1997), CHO cells (Strange et al., 2000) and in Xenopus oocytes (Strange et al., 2000; Song et al., 2002; Gagnon et al., 2006a). In contrast to KCC2, several studies revealed no significant cotransporter activity of KCC4 under isotonic conditions, but found that KCC4 could be strongly (about 20-fold) activated by hypotonic swelling (Mercado et al., 2000; Song et al., 2002). To identify molecular mechanisms underlining this difference, Mercado and coauthors used Xenopus oocytes and multiple KCC2-KCC4 chimeric constructs, and found a C-terminal region unique to KCC2 (ISO-domain, amino acids 1021–1035) that is both necessary and sufficient for the isotonic KCC2 activity (Mercado et al., 2006). A recent study confirmed important role of ISO domain by illustrating in transfected cultured hippocampal neurons that KCC2 mutant with deleted ISO domain showed reduced ion transport activity, whereas insertion of the ISO domain into KCC4 transporter, which is inactive under isotonic conditions, rendered it fully operational (Acton et al., 2012). Of notice, by using different KCC2-KCC4 chimera constructs overexpressed in Xenopus oocytes, Bergeron et al. (2006) obtained almost identical to Mercado et al. (2006) results showing that the deletion of distal KCC2 region strongly reduced KCC2 activity under isotonic conditions, whereas insertion of this domain to KCC4 potently activated transporter. However, after analysis of many other KCC2 and KCC4 mutants authors were not convinced that the ISO segment is “a prerequisite for K⁺-Cl⁻ cotransport to occur under isotonic conditions.” Clearly, a characterization of the mechanism of ISO domain action is one more challenge in the KCC2 field.

Taken together, the above data suggest that both N- and C-terminal domains of KCC might be directly involved in the control of the ion-transport unit; however, the mechanisms of their regulatory actions are not known. An important task for future studies will be to answer these questions by analysis of the three-dimensional structure of KCC proteins.

At least three trophic (growth) factors have been identified as efficient modulators of KCC2 functional expression: BDNF, Insulin-Like Growth Factor 1 (IGF-1), and Neurturin (Kelsch et al., 2001; Ludwig et al., 2011a; Shulga et al., 2012). Among them, the regulatory effects of BDNF on KCC2 are the most thoroughly characterized.

First evidence for the regulatory action of BDNF in developing neurons was presented by Aguado et al. (2003), who showed that in vivo transgenic over-expression of BDNF strongly increases KCC2 mRNA. Later the deletion of the receptor TrkB was shown to strongly decrease KCC2 mRNA (Carmona et al., 2006). Whether these effects are observed at the protein expression levels have so far not been reported in the literature. Consistent with this study, the application of BDNF to immature hippocampal cultures significantly increases the expression of KCC2 protein through Egr4-dependent KCC2 transcription (Ludwig et al., 2011b). Interestingly, in more mature cultures BDNF produced a decrease in KCC2 expression, thus revealing that the action of BDNF depends on the maturational state of neurons (see for review (Ferrini and De Koninck, 2013). So far, there is no evidence describing the regulatory action of the BDNF on the post-translational level at early stages of neuronal development.

In contrast to immature neurons, the exposure of adult neurons to BDNF in vivo and in vitro decreases KCC2 expression and function. BDNF decreases KCC2 mRNA levels (Rivera et al., 2002; Shulga et al., 2008), total KCC2 protein expression (Rivera et al., 2004), as well as tyrosine-phosphorylated (Wake et al., 2007) and membrane-inserted (Boulenguez et al., 2010) fractions of KCC2. Consistent with these findings, over-expression of BDNF in vivo produced a depolarizing shift of the GABA reversal potential in dorsal spinal cord lamina I (L1) neurons, presumably reflecting a decrease in KCC2 function (Coull et al., 2003, 2005).

Therefore, in mature neurons, application of exogenous BDNF induces a down-regulation of the KCC2 function. Endogenously released BDNF may therefore be a good candidate to mediate the activity-dependent down-regulation of KCC2. In support of this hypothesis, Rivera et al. (2004) showed that scavenging of endogenous BDNF, or inhibition of signaling downstream from BDNF TrkB receptors, effectively prevented activity-dependent reduction of KCC2 mRNA and protein. Several other studies have also reported a critical role of BDNF in the change of the inhibitory strength of GABA following axonal injury (Coull et al., 2005; Shulga et al., 2008; Boulenguez et al., 2010; Zhang et al., 2013). Intriguingly, BDNF application on neurons after injury, which are characterized by a decreased level of KCC2 expression, produced effects opposite to those observed in naive tissue, namely up-regulation of KCC2 surface expression (Shulga et al., 2008; Boulenguez et al., 2010). One plausible explanation is that injured neurons acquire properties of immature ones in order to launch a repair program (Kahle et al., 2008; Shulga et al., 2008, 2012).

Both during development (Westerholz et al., 2013) and in axotomized tissue (Shulga et al., 2009; Shulga and Rivera, 2013), the regulatory action of BDNF is tightly related to the thyroid hormone metabolism. The fact that thyroxine can bi-directionally regulate the expression of KCC2 in the same manner as BDNF would indicate that this effect could be mediated through BDNF signaling (Friauf et al., 2008; Shulga et al., 2009). Interestingly, the effect of thyroxin on KCC2 appears to be insensitive to BDNF signaling blockage indicating yet another pathway for regulation of KCC2 expression.

It has been long time appreciated that K⁺-Cl⁻ cotransport in non-excitable cells can be potently regulated by phosphorylation (Russell, 2000; Blaesse et al., 2009). Extrapolating these data to neuronal K⁺-Cl⁻ transport, several studies have made an attempt to characterize the role of protein phosphorylation in the functioning of the KCC2 (Kahle et al., 2013). Large number of putative KCC2 phosphorylation sites were shown to contribute into the control of transporter activity and/or its surface expression (Figure 1). Numerous studies have highlighted also different phosphorylation-related signaling pathways contributing to the control of KCC2 activity (Figure 3). Below we review the recent evidence and make an attempt to classify them with regard to the phosphorylation type (serine/threonine and tyrosine) and kinases implicated into the post-translational regulation of the KCC2. We emphasize, in particular, the discrepancies between different works and highlight unsolved questions.

Interesting data from groups of Hershfinkel and Aizenman (Hershfinkel et al., 2009; Chorin et al., 2011; Saadi et al., 2012) describe bi-directional Zn²⁺-dependent control of KCC2 (Figure 3). On one hand, intracellular Zn²⁺ in micromolar concentrations rapidly inhibits KCC2 activity (Hershfinkel et al., 2009). Although the mechanism of Zn²⁺ action is not clear, the finding indicates existence of a mechanism allowing inhibition of the intrinsic activity of KCC2. On the other hand, extracellular Zn²⁺ released from mossy fiber terminals, strongly activates KCC2 by increasing its surface expression (Chorin et al., 2011). Putatively, metabotropic zinc-sensing receptor (mZnR) is encoded by orphan Gq-coupled receptor (GPR39) and is coupled to phospholipase C (PLC)/extracellular-signal-regulated kinases (ERKs) pathway; the silencing of GPR39 or inhibition of PLC or ERKs abolished the Zn²⁺-dependent activation of KCC2 (Chorin et al., 2011). Consistent with a high rate of KCC2 turnover (Rivera et al., 2004), the authors have suggested that the increased surface expression of KCC2 is due to the inhibition of transporter internalization and/or degradation (Chorin et al., 2011). However, given recent evidence (see below chapter “Surface expression, turnover” and Puskarjov et al., 2012), one cannot exclude that Zn²⁺ potentiates the plasmalemmal insertion of the KCC2. The Zn²⁺-dependent control of KCC2 activity is one more subject in the KCC2 field requiring detailed analysis.

One more regulatory mechanism of KCC2 activity is through its direct interaction with other proteins (Figure 4). The first such interaction was described by Inoue et al. (2004) showing that brain-type creatine kinase (CKB) interacts with KCC2 and activates the transporter in HEK-293 cells (Inoue et al., 2004, 2006). Ikeda et al. (2004) found an intriguing interaction between the α2 subunit of the Na-K-ATPase pump and KCC2 that may stand for the breathing malfunction found in mice lacking the Na-K-ATPaseα2 subunit (Atp1a2^−/−). These mice display decreased functional activity of KCC2 and have abnormal spontaneous respiratory rhythm activity, similarly to the KCC2^−/− mice. KCC2 interacts also with other unrelated proteins such as the protein associated with Myc (PAM) via its RCC1 (Regulator of Chromatin Condensation) domain, resulting in the functional regulation of KCC2 activity in HEK-293 cells (Garbarini and Delpire, 2008), or with the cation-chloride cotransporter interacting protein 1 (CIP1) (Wenz et al., 2009), but the functional significance of this latter link in neurons remains unknown.

Furthermore, the cytoskeleton-associated protein 4.1N interacts with the cytoplasmic carboxy-terminus of KCC2 and is involved in dendritic spine formation (Li et al., 2007) and the plasticity of AMPA receptors (Gauvain et al., 2011). Interrupting the KCC2:4.1N interactions enhanced lateral diffusion of KCC2 away from excitatory synapses (Chamma et al., 2013). These results indicate a role for 4.1N in the local stabilization of KCC2 at the plasma membrane. Although lateral KCC2 diffusion was enhanced by NMDA receptor activity, the pathophysiological relevance of this is unknown.

Another molecule interacting with KCC2 is the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2) (Ivakine et al., 2013). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl⁻ extrusion, resulting in decreased synaptic inhibition in hippocampal neurons. Presently, the activity-dependent pathways controlling KCC2 via interaction with Neto2 are unknown, but consistent with Neto2 being part of a neuronal scaffolding platform (Tang et al., 2012), other studies will likely characterize the role of the KCC2:Neto2 interaction in neuronal plasticity or during excitotoxic events that affect the scaffold protein organization.

A recent finding describes a direct interaction between KCC2 and the beta isoform of the Rac/Cdc42 guanine nucleotide exchange factor (betaPIX). The synergetic action of KCC2 and betaPIX leads to decreased cofilin phosphorylation and change of glutamatergic synapse properties (Ludwig et al., 2013) but the importance of this interaction for activity-dependent signal transfer needs to be determined.

At least two studies have suggested an important role of the lipid rafts in the control of KCC2 activity (Hartmann et al., 2009; Watanabe et al., 2009). Lipid rafts are specialized membrane microdomains, enriched in glycosphingolipids and glycoproteins, serving as organising centers for the assembly of signaling molecules and control of their activity (for recent review see Lingwood and Simons, 2010). Both, Hartmann et al. (2009) and Watanabe et al. (2009) illustrated that in neurons approximately 50% of KCC2 is associated with lipid raft markers, whereas remaining transporter molecules are linked to non-rafts markers. However, author's conclusions on the importance of the function of the rafts-like organization for KCC2 were opposite: Hartmann et al. (2009) claimed that, when in rafts, KCC2 is in an inactive form, whereas Watanabe et al. (2009) were convinced that functional KCC2 forms rafts-like clusters. These contradictory assumptions, however, were made based on results obtained using different experimental paradigms, by employing different and wide-range acting pharmacological agents, and by exploring KCC2 mutants whose properties require more detailed analysis. Therefore, after careful and detailed critical analysis of both studies we propose that KCC2 might be membrane raft-associated protein, but additional experimental works are required from independent groups to clarify the role of lipid rafts in KCC2 functioning.

In summary, KCC2 is an important transporter controlling neuronal ion homeostasis and involved in several neurological disorders. The activity of this transporter could be rapidly modulated via large number of signaling pathways. The majority of these pathways remain to be characterized. In the Box 1, we review the existing methods used to study KCC2 activity in an attempt to help investigators in designing experiments to answer the many fundamental questions regarding KCC2 functional regulation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.