KCC2 is the neuron-specific K+-Cl(-) cotransporter required for maintaining low intracellular Cl(-), which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.

KCC2 is a neuron-specific K + -Cl − cotransporter that is essential for Cl − homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). 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. that makes fast GABA A Rmediated synaptic inhibition possible is maintained by the neuronspecific K + -Cl − cotransporter KCC2 (4), a member of the cationchloride cotransporter SLC12 gene family (5). KCC2 is essential for survival, as KCC2 knockout mice die immediately at birth due to respiratory failure (6). In the adult nervous system, decreased KCC2 expression correlates with neuropathic pain, spasticity following spinal cord injury, and epileptic seizures (5, 7-11).Based on the critical importance of KCC2 in the brain, an understanding of the mechanisms that promote and maintain KCC2 expression and efficacy is essential for designing therapeutic strategies to treat neurological disorders characterized by KCC2 dysfunction and the subsequent loss of inhibitory synaptic transmission. A major limitation in the development of these strategies is a lack of understanding regarding the cellular mechanisms regulating KCC2. In particular, KCC2 interacting proteins required for KCC2 transport and function in the mature CNS have not been identified, which represents a large gap in our fundamental knowledge regarding neuronal Cl − regulation and inhibitory synaptic transmission.Here, we identify neuropilin and tolloid like-2 (Neto2) as a KCC2 interacting protein in vivo and demonstrate that this interaction is required for normal neuronal Cl − homeostasis. Neto2 is a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein abundantly expressed in neurons (12), which is important for proper neurological function (13). CUB domains are evolutionarily conserved protein domains (14) that participate in protein-protein interactions (15, 16). In the present study, we performed an unbiased proteomic screen and discovered that Neto2 interacts with KCC2. We elucidated the role of this association and characterized its importance in the normal neurophysiological function of KCC2. Because Neto proteins were previously identified as auxiliary subunits of ionotropic glutamate receptors, including kainate and NMDA receptors (17-21), this study further extends the role of the Neto proteins to inhibitory synapses. ResultsNeto2 and KCC2 Interact in Vivo. Recent reports have demonstrated that Neto2 and its homologous protein Neto1 have important functions at excitatory synapses. Neto1 interacts with both the N-methyl D-aspartate receptor (NMDAR) and kainate receptor comp...

KCC2 is the neuron-specific member of the of K+-Cl− cotransporter gene family. It is also the only member of its family that is active under physiologically normal conditions, in the absence of osmotic stress. By extruding Cl− from the neuron under isotonic conditions, this transporter maintains a low concentration of neuronal Cl−, which is essential for fast inhibitory synaptic transmission by GABA and glycine in the mature nervous system. The other members of this K+-Cl− cotransporter gene family are exclusively swelling-activated. Here we demonstrate that a 15 amino acid region near the end of the C-terminus, unique to KCC2 (termed the ISO domain), is required for KCC2 to cotransport K+ and Cl− out of the neuron under isotonic conditions. We made this discovery by overexpressing chimeric KCC2-KCC4 cDNA constructs in cultured hippocampal neurons prepared from Sprague Dawley rat embryos and assaying neuronal Cl− through gramicidin perforated patch clamp recordings. We found that when neurons had been transfected with a chimeric KCC2 that lacked the unique ISO domain, hyperpolarizing responses to GABA were abolished. This finding indicates that the ISO domain is required for neuronal Cl− regulation. Furthermore, we discovered that when KCC2 lacks the ISO domain, it still retains swelling-activated transport, which demonstrates that there are exclusive molecular determinants of isotonic and swelling-induced K+-Cl− cotransport in neurons.

Coincident pre- and postsynaptic activity of hippocampal neurons alters the strength of gamma-aminobutyric acid (GABAA)-mediated inhibition through a Ca2+-dependent regulation of cation-chloride cotransporters. This long-term synaptic modulation is termed GABAergic spike-timing dependent plasticity (STDP). In the present study, we examined whether the properties of the GABAergic synapses themselves modulate the required postsynaptic Ca2+ influx during GABAergic STDP induction. To do this we first identified GABAergic synapses between cultured hippocampal neurons based on their relatively long decay time constants and their reversal potentials which lay close to the resting membrane potential. GABAergic STDP was then induced by coincidentally (±1 ms) firing the pre- and postsynaptic neurons at 5 Hz for 30 s, while postsynaptic Ca2+ was imaged with the Ca2+-sensitive fluorescent dye Fluo4-AM. In all cases, the induction of GABAergic STDP increased postsynaptic Ca2+ above resting levels. We further found that the magnitude of this increase correlated with the amplitude and polarity of the GABAergic postsynaptic current (GPSC); hyperpolarizing GPSCs reduced the Ca2+ influx in comparison to both depolarizing GPSCs, and postsynaptic neurons spiked alone. This relationship was influenced by both the driving force for Cl− and GABAA conductance (which had positive correlations with the Ca2+ influx). The spike-timing order during STDP induction did not influence the correlation between GPSC amplitude and Ca2+ influx, which is likely accounted for by the symmetrical GABAergic STDP window.

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