What biologists want from their chloride reporters - a conversation between chemists and biologists.

Impaired chloride transport affects diverse processes ranging from neuron excitability to water secretion, which underlie epilepsy and cystic fibrosis, respectively. The ability to image chloride fluxes with fluorescent probes has been essential for the investigation of the roles of chloride channels and transporters in health and disease. Therefore, developing effective fluorescent chloride reporters is critical to characterizing chloride transporters and discovering new ones. However, each chloride channel or transporter has a unique functional context that demands a suite of chloride probes with appropriate sensing characteristics. This Review seeks to juxtapose the biology of chloride transport with the chemistries underlying chloride sensors by exploring the various biological roles of chloride and highlighting the insights delivered by studies using chloride reporters. We then delineate the evolution of small-molecule sensors and genetically encoded chloride reporters. Finally, we analyze discussions with chloride biologists to identify the advantages and limitations of sensors in each biological context, as well as to recognize the key design challenges that must be overcome for developing the next generation of chloride sensors.

Restoring GABAergic inhibition rescues memory deficits in a Huntington's disease mouse model.

Huntington's disease (HD) is classically characterized as a movement disorder, however cognitive impairments precede the motor symptoms by ∼15 y. Based on proteomic and bioinformatic data linking the Huntingtin protein (Htt) and KCC2, which is required for hyperpolarizing GABAergic inhibition, and the important role of inhibition in learning and memory, we hypothesized that aberrant KCC2 function contributes to the hippocampal-associated learning and memory deficits in HD. We discovered that Htt and KCC2 interact in the hippocampi of wild-type and R6/2-HD mice, with a decrease in KCC2 expression in the hippocampus of R6/2 and YAC128 mice. The reduced expression of the Cl--extruding cotransporter KCC2 is accompanied by an increase in the Cl--importing cotransporter NKCC1, which together result in excitatory GABA in the hippocampi of HD mice. NKCC1 inhibition by the FDA-approved NKCC1 inhibitor bumetanide abolished the excitatory action of GABA and rescued the performance of R6/2 mice on hippocampal-associated behavioral tests.

Ionotropic and metabotropic kainate receptor signalling regulates Cl- homeostasis and GABAergic inhibition.

KEY POINTS: Potassium-chloride co-transporter 2 (KCC2) plays a critical role in regulating chloride homeostasis, which is essential for hyperpolarizing inhibition in the mature nervous system. KCC2 interacts with many proteins involved in excitatory neurotransmission, including the GluK2 subunit of the kainate receptor (KAR). We show that activation of KARs hyperpolarizes the reversal potential for GABA (EGABA ) via both ionotropic and metabotropic signalling mechanisms. KCC2 is required for the metabotropic KAR-mediated regulation of EGABA , although ionotropic KAR signalling can hyperpolarize EGABA independent of KCC2 transporter function. The KAR-mediated hyperpolarization of EGABA is absent in the GluK1/2-/- mouse and is independent of zinc release from mossy fibre terminals. The ability of KARs to regulate KCC2 function may have implications in diseases with disrupted excitation: inhibition balance, such as epilepsy, neuropathic pain, autism spectrum disorders and Down's syndrome. ABSTRACT: Potassium-chloride co-transporter 2 (KCC2) plays a critical role in the regulation of chloride (Cl- ) homeostasis within mature neurons. KCC2 is a secondarily active transporter that extrudes Cl- from the neuron, which maintains a low intracellular Cl- concentration [Cl- ]. This results in a hyperpolarized reversal potential of GABA (EGABA ), which is required for fast synaptic inhibition in the mature central nervous system. KCC2 also plays a structural role in dendritic spines and at excitatory synapses, and interacts with 'excitatory' proteins, including the GluK2 subunit of kainate receptors (KARs). KARs are glutamate receptors that display both ionotropic and metabotropic signalling. We show that activating KARs in the hippocampus hyperpolarizes EGABA , thus strengthening inhibition. This hyperpolarization occurs via both ionotropic and metabotropic KAR signalling in the CA3 region, whereas it is absent in the GluK1/2-/- mouse, and is independent of zinc release from mossy fibre terminals. The metabotropic signalling mechanism is dependent on KCC2, although the ionotropic signalling mechanism produces a hyperpolarization of EGABA even in the absence of KCC2 transporter function. These results demonstrate a novel functional interaction between a glutamate receptor and KCC2, a transporter critical for maintaining inhibition, suggesting that the KAR:KCC2 complex may play an important role in excitatory:inhibitory balance in the hippocampus. Additionally, the ability of KARs to regulate chloride homeostasis independently of KCC2 suggests that KAR signalling can regulate inhibition via multiple mechanisms. Activation of kainate-type glutamate receptors could serve as an important mechanism for increasing the strength of inhibition during periods of strong glutamatergic activity.

Emerging Mechanisms Underlying Dynamics of GABAergic Synapses.

Inhibitory circuits are diverse, yet with a poorly understood cell biology. Functional characterization of distinct inhibitory neuron subtypes has not been sufficient to explain how GABAergic neurotransmission sculpts principal cell activity in a relevant fashion. Our Mini-Symposium brings together several emerging mechanisms that modulate GABAergic neurotransmission dynamically from either the presynaptic or the postsynaptic site. The first two talks discuss novel developmental and neuronal subtype-specific contributions to the excitatory/inhibitory balance and circuit maturation. The next three talks examine how interactions between cellular pathways, lateral diffusion of proteins between synapses, and chloride transporter function at excitatory and inhibitory synapses and facilitate inhibitory synapse adaptations. Finally, we address functional differences within GABAergic interneurons to highlight the importance of diverse, flexible, and versatile inputs that shape network function. Together, the selection of topics demonstrates how developmental and activity-dependent mechanisms coordinate inhibition in relation to the excitatory inputs and vice versa.

A kainate receptor subunit promotes the recycling of the neuron-specific K+-Cl- co-transporter KCC2 in hippocampal neurons.

Synaptic inhibition depends on a transmembrane gradient of chloride, which is set by the neuron-specific K+-Cl- co-transporter KCC2. Reduced KCC2 levels in the neuronal membrane contribute to the generation of epilepsy, neuropathic pain, and autism spectrum disorders; thus, it is important to characterize the mechanisms regulating KCC2 expression. In the present study, we determined the role of KCC2-protein interactions in regulating total and surface membrane KCC2 expression. Using quantitative immunofluorescence in cultured mouse hippocampal neurons, we discovered that the kainate receptor subunit GluK2 and the auxiliary subunit Neto2 significantly increase the total KCC2 abundance in neurons but that GluK2 exclusively increases the abundance of KCC2 in the surface membrane. Using a live cell imaging assay, we further determined that KCC2 recycling primarily occurs within 1-2 h and that GluK2 produces an ∼40% increase in the amount of KCC2 recycled to the membrane during this time period. This GluK2-mediated increase in surface recycling translated to a significant increase in KCC2 expression in the surface membrane. Moreover, we found that KCC2 recycling is enhanced by protein kinase C-mediated phosphorylation of the GluK2 C-terminal residues Ser-846 and Ser-868. Lastly, using gramicidin-perforated patch clamp recordings, we found that the GluK2-mediated increase in KCC2 recycling to the surface membrane translates to a hyperpolarization of the reversal potential for GABA (EGABA). In conclusion, our results have revealed a mechanism by which kainate receptors regulate KCC2 expression in the hippocampus.

Regulation of neuronal chloride homeostasis by neuromodulators.

KCC2 is the central regulator of neuronal Cl(-) homeostasis, and is critical for enabling strong hyperpolarizing synaptic inhibition in the mature brain. KCC2 hypofunction results in decreased inhibition and increased network hyperexcitability that underlies numerous disease states including epilepsy, neuropathic pain and neuropsychiatric disorders. The current holy grail of KCC2 biology is to identify how we can rescue KCC2 hypofunction in order to restore physiological levels of synaptic inhibition and neuronal network activity. It is becoming increasingly clear that diverse cellular signals regulate KCC2 surface expression and function including neurotransmitters and neuromodulators. In the present review we explore the existing evidence that G-protein-coupled receptor (GPCR) signalling can regulate KCC2 activity in numerous regions of the nervous system including the hypothalamus, hippocampus and spinal cord. We present key evidence from the literature suggesting that GPCR signalling is a conserved mechanism for regulating chloride homeostasis. This evidence includes: (1) the activation of group 1 metabotropic glutamate receptors and metabotropic Zn(2+) receptors strengthens GABAergic inhibition in CA3 pyramidal neurons through a regulation of KCC2; (2) activation of the 5-hydroxytryptamine type 2A serotonin receptors upregulates KCC2 cell surface expression and function, restores endogenous inhibition in motoneurons, and reduces spasticity in rats; and (3) activation of A3A-type adenosine receptors rescues KCC2 dysfunction and reverses allodynia in a model of neuropathic pain. We propose that GPCR-signals are novel endogenous Cl(-) extrusion enhancers that may regulate KCC2 function.

Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons.

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.

Parvalbumin and GAD65 interneuron inhibition in the ventral hippocampus induces distinct behavioral deficits relevant to schizophrenia.

Hyperactivity within the ventral hippocampus (vHPC) has been linked to both psychosis in humans and behavioral deficits in animal models of schizophrenia. A local decrease in GABA-mediated inhibition, particularly involving parvalbumin (PV)-expressing GABA neurons, has been proposed as a key mechanism underlying this hyperactive state. However, direct evidence is lacking for a causal role of vHPC GABA neurons in behaviors associated with schizophrenia. Here, we probed the behavioral function of two different but overlapping populations of vHPC GABA neurons that express either PV or GAD65 by selectively inhibiting these neurons with the pharmacogenetic neuromodulator hM4D. We show that acute inhibition of vHPC GABA neurons in adult mice results in behavioral changes relevant to schizophrenia. Inhibiting either PV or GAD65 neurons produced distinct behavioral deficits. Inhibition of PV neurons, affecting ∼80% of the PV neuron population, robustly impaired prepulse inhibition of the acoustic startle reflex (PPI), startle reactivity, and spontaneous alternation, but did not affect locomotor activity. In contrast, inhibiting a heterogeneous population of GAD65 neurons, affecting ∼40% of PV neurons and 65% of cholecystokinin neurons, increased spontaneous and amphetamine-induced locomotor activity and reduced spontaneous alternation, but did not alter PPI. Inhibition of PV or GAD65 neurons also produced distinct changes in network oscillatory activity in the vHPC in vivo. Together, these findings establish a causal role for vHPC GABA neurons in controlling behaviors relevant to schizophrenia and suggest a functional dissociation between the GABAergic mechanisms involved in hippocampal modulation of sensorimotor processes.

Divergent opioid-mediated suppression of inhibition between hippocampus and neocortex across species and development.

Opioid receptors within the CNS regulate pain sensation and mood and are key targets for drugs of abuse. Within the adult rodent hippocampus (HPC), µ-opioid receptor agonists suppress inhibitory parvalbumin-expressing interneurons (PV-INs), thus disinhibiting the circuit. However, it is uncertain if this disinhibitory motif is conserved in other cortical regions, species, or across development. We observed that PV-IN mediated inhibition is robustly suppressed by opioids in HPC but not neocortex in mice and nonhuman primates, with spontaneous inhibitory tone in resected human tissue also following a consistent dichotomy. This hippocampal disinhibitory motif was established in early development when immature PV-INs and opioids already influence primordial network rhythmogenesis. Acute opioid-mediated modulation was partially occluded with morphine pretreatment, with implications for the effects of opioids on hippocampal network activity during circuit maturation as well as learning and memory. Together, these findings demonstrate that PV-INs exhibit a divergence in opioid sensitivity across brain regions that is remarkably conserved across evolution and highlights the underappreciated role of opioids acting through immature PV-INs in shaping hippocampal development.

Increasing CRTC1 function in the dentate gyrus during memory formation or reactivation increases memory strength without compromising memory quality.

Memory stabilization following encoding (synaptic consolidation) or memory reactivation (reconsolidation) requires gene expression and protein synthesis (Dudai and Eisenberg, 2004; Tronson and Taylor, 2007; Nader and Einarsson, 2010; Alberini, 2011). Although consolidation and reconsolidation may be mediated by distinct molecular mechanisms (Lee et al., 2004), disrupting the function of the transcription factor CREB impairs both processes (Kida et al., 2002; Mamiya et al., 2009). Phosphorylation of CREB at Ser133 recruits CREB binding protein (CBP)/p300 coactivators to activate transcription (Chrivia et al., 1993; Parker et al., 1996). In addition to this well known mechanism, CREB regulated transcription coactivators (CRTCs), previously called transducers of regulated CREB (TORC) activity, stimulate CREB-mediated transcription, even in the absence of CREB phosphorylation. Recently, CRTC1 has been shown to undergo activity-dependent trafficking from synapses and dendrites to the nucleus in excitatory hippocampal neurons (Ch'ng et al., 2012). Despite being a powerful and specific coactivator of CREB, the role of CRTC in memory is virtually unexplored. To examine the effects of increasing CRTC levels, we used viral vectors to locally and acutely increase CRTC1 in the dorsal hippocampus dentate gyrus region of mice before training or memory reactivation in context fear conditioning. Overexpressing CRTC1 enhanced both memory consolidation and reconsolidation; CRTC1-mediated memory facilitation was context specific (did not generalize to nontrained context) and long lasting (observed after virally expressed CRTC1 dissipated). CREB overexpression produced strikingly similar effects. Therefore, increasing CRTC1 or CREB function is sufficient to enhance the strength of new, as well as established reactivated, memories without compromising memory quality.

Hyperpolarizing GABAergic transmission requires the KCC2 C-terminal ISO domain.

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 aa 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.

NMDARs Drive the Expression of Neuropsychiatric Disorder Risk Genes Within GABAergic Interneuron Subtypes in the Juvenile Brain.

Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical, and physiological properties. However, the molecular mechanisms regulating their maturation remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit gene Grin1 mediates transcriptional regulation of gene expression in specific subtypes of MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific early developmental Grin1 loss results in a broad downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs in the juvenile brain. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

Translatome Analyses Using Conditional Ribosomal Tagging in GABAergic Interneurons and Other Sparse Cell Types.

GABAergic interneurons comprise a small but diverse subset of neurons in the mammalian brain that tightly regulate neuronal circuit maturation and information flow and, ultimately, behavior. Because of their centrality in the etiology of numerous neurological disorders, examining the molecular architecture of these neurons under different physiological scenarios has piqued the interest of the broader neuroscience community. The last few years have seen an explosion in next-generation sequencing (NGS) approaches aimed at identifying genetic and state-dependent subtypes in neuronal diversity. Although several approaches are employed to address neuronal molecular diversity, ribosomal tagging has emerged at the forefront of identifying the translatomes of neuronal subtypes. This approach primarily relies on Cre recombinase-driven expression of hemagglutinin A (HA)-tagged RiboTag mice exclusively in the neuronal subtype of interest. This allows the immunoprecipitation of cell-type-specific, ribosome-engaged mRNA, expressed both in the soma and the neuronal processes, for targeted quantitative real-time PCR (qRT-PCR) or high-throughput RNA sequencing analyses. Here we detail the typical technical caveats associated with successful application of the RiboTag technique for analyzing GABAergic interneurons, and in theory other sparse cell types, in the central nervous system. Published 2020. U.S. Government. Basic Protocol 1: Breeding mice to obtain RiboTag homozygosity Support Protocol 1: Detection of ectopic Cre recombinase expression Basic Protocol 2: The RiboTag assay Support Protocol 2: Real-time quantitative PCR (qRT-PCR) assay of RiboTag-derived cell-type-specific RNA Support Protocol 3: Construction of cell-type-specific RNA-seq library Support Protocol 4: Secondary analyses of RiboTag-derived RNA-seq dataset.

Emergence of non-canonical parvalbumin-containing interneurons in hippocampus of a murine model of type I lissencephaly.

Type I lissencephaly is a neuronal migration disorder caused by haploinsuffiency of the PAFAH1B1 (mouse: Pafah1b1) gene and is characterized by brain malformation, developmental delays, and epilepsy. Here, we investigate the impact of Pafah1b1 mutation on the cellular migration, morphophysiology, microcircuitry, and transcriptomics of mouse hippocampal CA1 parvalbumin-containing inhibitory interneurons (PV+INTs). We find that WT PV+INTs consist of two physiological subtypes (80% fast-spiking (FS), 20% non-fast-spiking (NFS)) and four morphological subtypes. We find that cell-autonomous mutations within interneurons disrupts morphophysiological development of PV+INTs and results in the emergence of a non-canonical 'intermediate spiking (IS)' subset of PV+INTs. We also find that now dominant IS/NFS cells are prone to entering depolarization block, causing them to temporarily lose the ability to initiate action potentials and control network excitation, potentially promoting seizures. Finally, single-cell nuclear RNAsequencing of PV+INTs revealed several misregulated genes related to morphogenesis, cellular excitability, and synapse formation.

Activity-dependent tuning of intrinsic excitability in mouse and human neurogliaform cells.

The ability to modulate the efficacy of synaptic communication between neurons constitutes an essential property critical for normal brain function. Animal models have proved invaluable in revealing a wealth of diverse cellular mechanisms underlying varied plasticity modes. However, to what extent these processes are mirrored in humans is largely uncharted thus questioning their relevance in human circuit function. In this study, we focus on neurogliaform cells, that possess specialized physiological features enabling them to impart a widespread inhibitory influence on neural activity. We demonstrate that this prominent neuronal subtype, embedded in both mouse and human neural circuits, undergo remarkably similar activity-dependent modulation manifesting as epochs of enhanced intrinsic excitability. In principle, these evolutionary conserved plasticity routes likely tune the extent of neurogliaform cell mediated inhibition thus constituting canonical circuit mechanisms underlying human cognitive processing and behavior.

Depolarizing GABA Transmission Restrains Activity-Dependent Glutamatergic Synapse Formation in the Developing Hippocampal Circuit.

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mature brain but has the paradoxical property of depolarizing neurons during early development. Depolarization provided by GABAA transmission during this early phase regulates neural stem cell proliferation, neural migration, neurite outgrowth, synapse formation, and circuit refinement, making GABA a key factor in neural circuit development. Importantly, depending on the context, depolarizing GABAA transmission can either drive neural activity or inhibit it through shunting inhibition. The varying roles of depolarizing GABAA transmission during development, and its ability to both drive and inhibit neural activity, makes it a difficult developmental cue to study. This is particularly true in the later stages of development when the majority of synapses form and GABAA transmission switches from depolarizing to hyperpolarizing. Here, we addressed the importance of depolarizing but inhibitory (or shunting) GABAA transmission in glutamatergic synapse formation in hippocampal CA1 pyramidal neurons. We first showed that the developmental depolarizing-to-hyperpolarizing switch in GABAA transmission is recapitulated in organotypic hippocampal slice cultures. Based on the expression profile of K+-Cl- co-transporter 2 (KCC2) and changes in the GABA reversal potential, we pinpointed the timing of the switch from depolarizing to hyperpolarizing GABAA transmission in CA1 neurons. We found that blocking depolarizing but shunting GABAA transmission increased excitatory synapse number and strength, indicating that depolarizing GABAA transmission can restrain glutamatergic synapse formation. The increase in glutamatergic synapses was activity-dependent but independent of BDNF signaling. Importantly, the elevated number of synapses was stable for more than a week after GABAA inhibitors were washed out. Together these findings point to the ability of immature GABAergic transmission to restrain glutamatergic synapse formation and suggest an unexpected role for depolarizing GABAA transmission in shaping excitatory connectivity during neural circuit development.

Neocortical Projection Neurons Instruct Inhibitory Interneuron Circuit Development in a Lineage-Dependent Manner.

Neocortical circuits consist of stereotypical motifs that must self-assemble during development. Recent evidence suggests that the subtype identity of both excitatory projection neurons (PNs) and inhibitory interneurons (INs) is important for this process. We knocked out the transcription factor Satb2 in PNs to induce those of the intratelencephalic (IT) type to adopt a pyramidal tract (PT)-type identity. Loss of IT-type PNs selectively disrupted the lamination and circuit integration of INs derived from the caudal ganglionic eminence (CGE). Strikingly, reprogrammed PNs demonstrated reduced synaptic targeting of CGE-derived INs relative to controls. In control mice, IT-type PNs targeted neighboring CGE INs, while PT-type PNs did not in deep layers, confirming this lineage-dependent motif. Finally, single-cell RNA sequencing revealed that major CGE IN subtypes were conserved after loss of IT PNs, but with differential transcription of synaptic proteins and signaling molecules. Thus, IT-type PNs influence CGE-derived INs in a non-cell-autonomous manner during cortical development.

Neto Auxiliary Subunits Regulate Interneuron Somatodendritic and Presynaptic Kainate Receptors to Control Network Inhibition.

Although Netos are considered auxiliary subunits critical for kainate receptor (KAR) function, direct evidence for their regulation of native KARs is limited. Because Neto KAR regulation is GluK subunit/Neto isoform specific, such regulation must be determined in cell-type-specific contexts. We demonstrate Neto1/2 expression in somatostatin (SOM)-, cholecystokinin/cannabinoid receptor 1 (CCK/CB1)-, and parvalbumin (PV)-containing interneurons. KAR-mediated excitation of these interneurons is contingent upon Neto1 because kainate yields comparable effects in Neto2 knockouts and wild-types but fails to excite interneurons or recruit inhibition in Neto1 knockouts. In contrast, presynaptic KARs in CCK/CB1 interneurons are dually regulated by both Neto1 and Neto2. Neto association promotes tonic presynaptic KAR activation, dampening CCK/CB1 interneuron output, and loss of this brake in Neto mutants profoundly increases CCK/CB1 interneuron-mediated inhibition. Our results confirm that Neto1 regulates endogenous somatodendritic KARs in diverse interneurons and demonstrate Neto regulation of presynaptic KARs in mature inhibitory presynaptic terminals.

Native KCC2 interactome reveals PACSIN1 as a critical regulator of synaptic inhibition.

KCC2 is a neuron-specific K+-Cl- cotransporter essential for establishing the Cl- gradient required for hyperpolarizing inhibition in the central nervous system (CNS). KCC2 is highly localized to excitatory synapses where it regulates spine morphogenesis and AMPA receptor confinement. Aberrant KCC2 function contributes to human neurological disorders including epilepsy and neuropathic pain. Using functional proteomics, we identified the KCC2-interactome in the mouse brain to determine KCC2-protein interactions that regulate KCC2 function. Our analysis revealed that KCC2 interacts with diverse proteins, and its most predominant interactors play important roles in postsynaptic receptor recycling. The most abundant KCC2 interactor is a neuronal endocytic regulatory protein termed PACSIN1 (SYNDAPIN1). We verified the PACSIN1-KCC2 interaction biochemically and demonstrated that shRNA knockdown of PACSIN1 in hippocampal neurons increases KCC2 expression and hyperpolarizes the reversal potential for Cl-. Overall, our global native-KCC2 interactome and subsequent characterization revealed PACSIN1 as a novel and potent negative regulator of KCC2.

Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures.

Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.