Fast Synaptic Inhibition Research Articles

All-optical reporting of inhibitory receptor driving force in the nervous system

Ionic driving forces provide the net electromotive force for ion movement across receptors, channels, and transporters, and are a fundamental property of all cells. In the nervous system, fast synaptic inhibition is mediated by chloride permeable GABAA and glycine receptors, and single-cell intracellular recordings have been the only method for estimating driving forces across these receptors (DFGABAA). Here we present a tool for quantifying inhibitory receptor driving force named ORCHID: all-Optical Reporting of CHloride Ion Driving force. We demonstrate ORCHID’s ability to provide accurate, high-throughput measurements of resting and dynamic DFGABAA from genetically targeted cell types over multiple timescales. ORCHID confirms theoretical predictions about the biophysical mechanisms that establish DFGABAA, reveals differences in DFGABAA between neurons and astrocytes, and affords the first in vivo measurements of intact DFGABAA. This work extends our understanding of inhibitory synaptic transmission and demonstrates the potential for all-optical methods to assess ionic driving forces.

Daily rhythm in cortical chloride homeostasis underpins functional changes in visual cortex excitability

Cortical activity patterns are strongly modulated by fast synaptic inhibition mediated through ionotropic, chloride-conducting receptors. Consequently, chloride homeostasis is ideally placed to regulate activity. We therefore investigated the stability of baseline [Cl-]i in adult mouse neocortex, using in vivo two-photon imaging. We found a two-fold increase in baseline [Cl-]i in layer 2/3 pyramidal neurons, from day to night, with marked effects upon both physiological cortical processing and seizure susceptibility. Importantly, the night-time activity can be converted to the day-time pattern by local inhibition of NKCC1, while inhibition of KCC2 converts day-time [Cl-]i towards night-time levels. Changes in the surface expression and phosphorylation of the cation-chloride cotransporters, NKCC1 and KCC2, matched these pharmacological effects. When we extended the dark period by 4 h, mice remained active, but [Cl-]i was modulated as for animals in normal light cycles. Our data thus demonstrate a daily [Cl-]i modulation with complex effects on cortical excitability.

Active cortical networks promote shunting fast synaptic inhibition in vivo

Fast synaptic inhibition determines neuronal response properties in the mammalian brain and is mediated by chloride-permeable ionotropic GABA-A receptors (GABAARs). Despite their fundamental role, it is still not known how GABAARs signal in the intact brain. Here, we use invivo gramicidin recordings to investigate synaptic GABAAR signaling in mouse cortical pyramidal neurons under conditions that preserve native transmembrane chloride gradients. In anesthetized cortex, synaptic GABAARs exert classic hyperpolarizing effects. In contrast, GABAAR-mediated synaptic signaling in awake cortex is found to be predominantly shunting. This is due to more depolarized GABAAR equilibrium potentials (EGABAAR), which are shown to result from the high levels of synaptic activity that characterize awake cortical networks. Synaptic EGABAAR observed in awake cortex facilitates the desynchronizing effects of inhibitory inputs upon local networks, which increases the flexibility of spiking responses to external inputs. Our findings therefore suggest that GABAAR signaling adapts to optimize cortical functions.

Spectrin-beta 2 facilitates the selective accumulation of GABAA receptors at somatodendritic synapses

Fast synaptic inhibition is dependent on targeting specific GABAAR subtypes to dendritic and axon initial segment (AIS) synapses. Synaptic GABAARs are typically assembled from α1-3, β and γ subunits. Here, we isolate distinct GABAARs from the brain and interrogate their composition using quantitative proteomics. We show that α2-containing receptors co-assemble with α1 subunits, whereas α1 receptors can form GABAARs with α1 as the sole α subunit. We demonstrate that α1 and α2 subunit-containing receptors co-purify with distinct spectrin isoforms; cytoskeletal proteins that link transmembrane proteins to the cytoskeleton. β2-spectrin was preferentially associated with α1-containing GABAARs at dendritic synapses, while β4-spectrin was associated with α2-containing GABAARs at AIS synapses. Ablating β2-spectrin expression reduced dendritic and AIS synapses containing α1 but increased the number of synapses containing α2, which altered phasic inhibition. Thus, we demonstrate a role for spectrins in the synapse-specific targeting of GABAARs, determining the efficacy of fast neuronal inhibition.

Pharmacological Signature and Target Specificity of Inhibitory Circuits Formed by Martinotti Cells in the Mouse Barrel Cortex.

In the neocortex, fast synaptic inhibition orchestrates both spontaneous and sensory-evoked activity. GABAergic interneurons (INs) inhibit pyramidal neurons (PNs) directly, modulating their output activity and thus contributing to balance cortical networks. Moreover, several IN subtypes also inhibit other INs, forming specific disinhibitory circuits, which play crucial roles in several cognitive functions. Here, we studied a subpopulation of somatostatin-positive INs, the Martinotti cells (MCs) in layer 2/3 of the mouse barrel cortex (both sexes). MCs inhibit the distal portion of PN apical dendrites, thus controlling dendrite electrogenesis and synaptic integration. Yet, it is poorly understood whether MCs inhibit other elements of the cortical circuits, and the connectivity properties with non-PN targets are unknown. We found that MCs have a strong preference for PN dendrites, but they also considerably connect with parvalbumin-positive, vasoactive intestinal peptide-expressing, and layer 1 (L1) INs. Remarkably, GABAergic synapses from MCs exhibited clear cell type-specific short-term plasticity. Moreover, whereas the biophysical properties of MC-PN synapses were consistent with distal dendritic inhibition, MC-IN synapses exhibited characteristics of fast perisomatic inhibition. Finally, MC-PN connections used α5-containing GABAA receptors (GABAARs), but this subunit was not expressed by the other INs targeted by MCs. We reveal a specialized connectivity blueprint of MCs within different elements of superficial cortical layers. In addition, our results identify α5-GABAARs as the molecular fingerprint of MC-PN dendritic inhibition. This is of critical importance, given the role of α5-GABAARs in cognitive performance and their involvement in several brain diseases.SIGNIFICANCE STATEMENT Martinotti cells (MCs) are a prominent, broad subclass of somatostatin-expressing GABAergic interneurons, specialized in controlling distal dendrites of pyramidal neurons (PNs) and taking part in several cognitive functions. Here we characterize the connectivity pattern of MCs with other interneurons in the superficial layers (L1 and L2/3) of the mouse barrel cortex. We found that the connectivity pattern of MCs with PNs as well as parvalbumin, vasoactive intestinal peptide, and L1 interneurons exhibit target-specific plasticity and biophysical properties. The specificity of α5-GABAARs at MC-PN synapses and the lack or functional expression of this subunit by other cell types define the molecular identity of MC-PN connections and the exclusive involvement of this inhibitory circuits in α5-dependent cognitive tasks.

Chronic, episodic developmental nicotine exposure alters GABAergic synaptic transmission to hypoglossal motor neurons at a critical developmental age in rats

The adverse effects of nicotine exposure on brain structural and functional development is attributed to its action on the nicotinic acetylcholine receptors (nAChRs). nAChRs are expressed post‐synaptically on hypoglossal motor neurons (XIIMNs), the neurons that innervate the tongue. We recently showed that chronic, episodic developmental nicotine exposure alters the intrinsic properties of these neurons, which may be one mechanism to explain impaired tongue muscle function that is commonly observed in nicotine‐ exposed human and animal neonates. Importantly, along with the intrinsic properties of XIIMNs, the balance of excitatory and inhibitory fast‐synaptic inputs to these neurons is important in dictating the strength of motor output to the tongue. nAChRs are also located on the soma, dendrites, and axon end terminals of excitatory and inhibitory interneurons that synapse onto XIIMNs, activation of which increases action potential firing and neurotransmitter release. Here, we tested the hypothesis that chronic, episodic developmental nicotine exposure alters GABAergic transmission, a major source of fast‐synaptic inhibition, to XIIMNs. XIIMNs were recorded in brainstem slices from control and nicotine‐ exposed rats in two age groups: postnatal days (P)1‐5 and P10‐12, which is considered a critical period of brain development. In voltage‐ clamp mode, the frequency of GABAergic spontaneous inhibitory post‐synaptic currents (sIPSCs) was recorded at baseline and in response to activation of nAChRs with acute, bath‐applied nicotine. The baseline frequency of GABAergic sIPSCs was not different between control and nicotine‐exposed neurons at either age. In the P1‐5 age group, acute nicotine increased the frequency of GABAergic sIPSCs in both control and nicotine‐exposed neurons. However, at ages P10‐12, acute nicotine increased the frequency of GABAergic sIPSCs in nicotine‐exposed neurons, but had no effect in control neurons. These findings indicate that chronic, episodic nicotine exposure alters GABAergic synaptic transmission to XIIMNs at a critical developmental age, and may contribute to excitatory/inhibitory imbalance that underlies impaired control of the muscles of the tongue.

Striatal Chloride Dysregulation and Impaired GABAergic Signaling Due to Cation-Chloride Cotransporter Dysfunction in Huntington's Disease.

Intracellular chloride (Cl–) levels in mature neurons must be tightly regulated for the maintenance of fast synaptic inhibition. In the mature central nervous system (CNS), synaptic inhibition is primarily mediated by gamma-amino butyric acid (GABA), which binds to Cl– permeable GABAA receptors (GABAARs). The intracellular Cl– concentration is primarily maintained by the antagonistic actions of two cation-chloride cotransporters (CCCs): Cl–-importing Na+-K+-Cl– co-transporter-1 (NKCC1) and Cl– -exporting K+-Cl– co-transporter-2 (KCC2). In mature neurons in the healthy brain, KCC2 expression is higher than NKCC1, leading to lower levels of intracellular Cl–, and Cl– influx upon GABAAR activation. However, in neurons of the immature brain or in neurological disorders such as epilepsy and traumatic brain injury, impaired KCC2 function and/or enhanced NKCC1 expression lead to intracellular Cl– accumulation and GABA-mediated excitation. In Huntington’s disease (HD), KCC2- and NKCC1-mediated Cl–-regulation are also altered, which leads to GABA-mediated excitation and contributes to the development of cognitive and motor impairments. This review summarizes the role of Cl– (dys)regulation in the healthy and HD brain, with a focus on the basal ganglia (BG) circuitry and CCCs as potential therapeutic targets in the treatment of HD.

Gephyrin Interacts with the K-Cl Cotransporter KCC2 to Regulate Its Surface Expression and Function in Cortical Neurons.

The K+-Cl- cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAA receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABAA receptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling.

Activation of GABA(A) receptors inhibits T cell proliferation.

BackgroundThe major sites for fast synaptic inhibition in the central nervous system (CNS) are ion channels activated by γ-aminobutyric acid (GABA). These receptors are referred as GABA(A) receptors (GABA(A)R). Recent evidence indicates a role of GABA(A)R in modulating the immune response. This work aimed to discern the role of GABA and GABA(A)Rs in human and mouse T cell activity.MethodsMouse splenocytes or human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 antibodies and the proliferation of both CD8+ and CD4+ T cells assessed through flow cytometry. Subsequently, the effects on T cell proliferation of either GABA(A)R modulation by diazepam that is also capable of activating mitochondrial based translocator protein (TSPO), alprazolam and allopregnanolone or inhibition by bicucculine methiodide (BMI) and (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) were assessed.ResultsPositive modulation of GABA(A)Rs either by benzodiazepines or the neurosteroid allopregnanolone inhibits both mouse and human T cell proliferation. GABAergic inhibition of T cell proliferation by benzodiazepines could be rescued by GABA(A)R blocking. Our data suggest that benzodiazepines influence T cell proliferation through both TSPO and GABA(A)Rs activation.ConclusionsWe conclude that activation of GABA(A)Rs provides immunosuppression by inhibiting T cell proliferation.

Enkephalin release from VIP interneurons in the hippocampal CA2/3a region mediates heterosynaptic plasticity and social memory.

The hippocampus contains a diverse array of inhibitory interneurons that gate information flow through local cortico-hippocampal circuits to regulate memory storage. Although most studies of interneurons have focused on their role in fast synaptic inhibition mediated by GABA release, different classes of interneurons express unique sets of neuropeptides, many of which have been shown to exert powerful effects on neuronal function and memory when applied pharmacologically. However, relatively little is known about whether and how release of endogenous neuropeptides from inhibitory cells contributes to their behavioral role in regulating memory formation. Here we report that vasoactive intestinal peptide (VIP)-expressing interneurons participate in social memory storage by enhancing information transfer from hippocampal CA3 pyramidal neurons to CA2 pyramidal neurons. Notably, this action depends on release of the neuropeptide enkephalin from VIP neurons, causing long-term depression of feedforward inhibition onto CA2 pyramidal cells. Moreover, VIP neuron activity in the CA2 region is increased selectively during exploration of a novel conspecific. Our findings, thus, enhance our appreciation of how GABAergic neurons can regulate synaptic plasticity and mnemonic behavior by demonstrating that such actions can be mediated by release of a specific neuropeptide, rather than through classic fast inhibitory transmission.

Modulation of recombinant human alpha 1 glycine receptor by flavonoids and gingerols.

The inhibitory glycine receptor (GlyR) is a principal mediator of fast synaptic inhibition in mammalian spinal cord, brainstem, and higher brain centres. Flavonoids are secondary plant metabolites that exhibit many beneficial physiological effects, including modulatory action on neuronal receptors. Using whole-cell current recordings from recombinant human α1 GlyRs, expressed in HEK293cells, we compared the flavonols kaempferol and quercetin, the flavanone naringenin, the flavones apigenin and nobiletin, the isoflavone genistein, and two gingerols, 6-gingerol and 8-gingerol for their modulation of receptor currents. All compounds were inhibitors of the GlyR with IC50 values ranging between 9.3±2.6µM (kaempferol) and 46.7±6.5µM (genistein), following a mixed mode of inhibition. Co-application of two inhibitors revealed distinct binding sites for flavonoids and gingerols. Pore-lining mutants T258A and T258S were strongly inhibited by quercetin and naringenin, but not by 6-gingerol, confirming the existence of distinct binding sites for flavonoids and gingerols. Apigenin, kaempferol, nobiletin, naringenin and 6-gingerol showed biphasic action, potentiating glycine-induced currents at low concentration of both, modulator and glycine, and inhibiting at higher concentrations. Identification of distinct modulatory sites for flavonoids and related compounds may present pharmacological target sites and aid the discovery of novel glycinergic drugs.

KCC2 is required for the survival of mature neurons but not for their development

The K+/Cl− cotransporter KCC2 (SLC12A5) allows mature neurons in the CNS to maintain low intracellular Cl− levels that are critical in mediating fast hyperpolarizing synaptic inhibition via type A γ-aminobutyric acid receptors (GABAARs). In accordance with this, compromised KCC2 activity results in seizures, but whether such deficits directly contribute to the subsequent changes in neuronal structure and viability that lead to epileptogenesis remains to be assessed. Canonical hyperpolarizing GABAAR currents develop postnatally, which reflect a progressive increase in KCC2 expression levels and activity. To investigate the role that KCC2 plays in regulating neuronal viability and architecture, we have conditionally ablated KCC2 expression in developing and mature neurons. Decreasing KCC2 expression in mature neurons resulted in the rapid activation of the extrinsic apoptotic pathway. Intriguingly, direct pharmacological inhibition of KCC2 in mature neurons was sufficient to rapidly induce apoptosis, an effect that was not abrogated via blockade of neuronal depolarization using tetrodotoxin (TTX). In contrast, ablating KCC2 expression in immature neurons had no discernable effects on their subsequent development, arborization, or dendritic structure. However, removing KCC2 in immature neurons was sufficient to ablate the subsequent postnatal development of hyperpolarizing GABAAR currents. Collectively, our results demonstrate that KCC2 plays a critical role in neuronal survival by limiting apoptosis, and mature neurons are highly sensitive to the loss of KCC2 function. In contrast, KCC2 appears to play a minimal role in mediating neuronal development or architecture.

A proline-rich motif in the large intracellular loop of the glycine receptor α1 subunit interacts with the Pleckstrin homology domain of collybistin

IntroductionThe inhibitory glycine receptor (GlyR), a mediator of fast synaptic inhibition, is located and held at neuronal synapses through the anchoring proteins gephyrin and collybistin. Stable localization of neurotransmitter receptors is essential for synaptic function. In case of GlyRs, only beta subunits were known until now to mediate synaptic anchoring. ObjectivesWe identified a poly-proline II helix (PPII) in position 365–373 of the intra-cellular TM3-4 loop of the human GlyRα1 subunit as a novel potential synaptic anchoring site. The potential role of the PPII helix as synaptic anchoring site was tested. MethodsGlycine receptors and collybistin variants were generated and recombinantly expressed in HEK293 cells and cultured neurons. Receptor function was assessed using patch-clamp electrophysiology, protein-protein interaction was studied using co-immuno-precipitation and pulldown experiments. ResultsRecombinantly expressed collybistin bound to isolated GlyRα1 TM3-4 loops in GST-pulldown assays. When the five proline residues P365A, P366A, P367A, P369A, P373A (GlyRα1P1-5A) located in the GlyRα1-PPII helix were replaced by alanines, the PPII secondary structure was disrupted. Recombinant GlyRα1P1-5A mutant subunits displayed normal cell surface expression and wildtype-like ion channel function, but binding to collybistin was abolished. The GlyRα1-collybistin interaction was independently confirmed by o-immunoprecipitation assays using full-length GlyRα1 subunits. Surprisingly, the interaction was not mediated by the SH3 domain of collybistin, but by its Pleckstrin homology (PH) domain. The mutation GlyRα1P366L, identified in a hyperekplexia patient, is also disrupting the PPII helix, and caused reduced collybistin binding. ConclusionOur data suggest a novel interaction between α1 GlyR subunits and collybistin, which is physiologically relevant in vitro and in vivo and may contribute to postsynaptic anchoring of glycine receptors.

Phosphorylation on Ser-359 of the α2 subunit in GABA type A receptors down-regulates their density at inhibitory synapses

GABA type A receptors (GABAARs) mediate fast synaptic inhibition and are trafficked to functionally diverse synapses. However, the precise molecular mechanisms that regulate the synaptic targeting of these receptors are unclear. Whereas it has been previously shown that phosphorylation events in α4, β, and γ subunits of GABAARs govern their function and trafficking, phosphorylation of other subunits has not yet been demonstrated. Here, we show that the α2 subunit of GABAARs is phosphorylated at Ser-359 and enables dynamic regulation of GABAAR binding to the scaffolding proteins gephyrin and collybistin. We initially identified Ser-359 phosphorylation by MS analysis, and additional experiments revealed that it is regulated by the activities of cAMP-dependent protein kinase (PKA) and the protein phosphatase 1 (PP1) and/or PP2A. GST-based pulldowns and coimmunoprecipitation experiments demonstrate preferential binding of both gephyrin and collybistin to WT and an S359A phosphonull variant, but not to an S359D phosphomimetic variant. Furthermore, the decreased capacity of the α2 S359D variant to bind collybistin and gephyrin decreased the density of synaptic α2-containing GABAAR clusters and caused an absence of α2 enrichment in the axon initial segment. These results suggest that PKA-mediated phosphorylation and PP1/PP2A-dependent dephosphorylation of the α2 subunit play a role in the dynamic regulation of GABAAR accumulation at inhibitory synapses, thereby regulating the strength of synaptic inhibition. The MS data have been deposited to ProteomeXchange, with the data set identifier PXD019597.

Chloride dynamics alter the input-output properties of neurons.

Fast synaptic inhibition is a critical determinant of neuronal output, with subcellular targeting of synaptic inhibition able to exert different transformations of the neuronal input-output function. At the receptor level, synaptic inhibition is primarily mediated by chloride-permeable Type A GABA receptors. Consequently, dynamics in the neuronal chloride concentration can alter the functional properties of inhibitory synapses. How differences in the spatial targeting of inhibitory synapses interact with intracellular chloride dynamics to modulate the input-output function of neurons is not well understood. To address this, we developed computational models of multi-compartment neurons that incorporate experimentally parametrised mechanisms to account for neuronal chloride influx, diffusion, and extrusion. We found that synaptic input (either excitatory, inhibitory, or both) can lead to subcellular variations in chloride concentration, despite a uniform distribution of chloride extrusion mechanisms. Accounting for chloride changes resulted in substantial alterations in the neuronal input-output function. This was particularly the case for peripherally targeted dendritic inhibition where dynamic chloride compromised the ability of inhibition to offset neuronal input-output curves. Our simulations revealed that progressive changes in chloride concentration mean that the neuronal input-output function is not static but varies significantly as a function of the duration of synaptic drive. Finally, we found that the observed effects of dynamic chloride on neuronal output were mediated by changes in the dendritic reversal potential for GABA. Our findings provide a framework for understanding the computational effects of chloride dynamics on dendritically targeted synaptic inhibition.

An axon-specific expression of HCN channels catalyzes fast action potential signaling in GABAergic interneurons

During high-frequency network activities, fast-spiking, parvalbumin-expressing basket cells (PV+-BCs) generate barrages of fast synaptic inhibition to control the probability and precise timing of action potential (AP) initiation in principal neurons. Here we describe a subcellular specialization that contributes to the high speed of synaptic inhibition mediated by PV+-BCs. Mapping of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution in rat hippocampal PV+-BCs with subcellular patch-clamp methods revealed that functional HCN channels are exclusively expressed in axons and completely absent from somata and dendrites. HCN channels not only enhance AP initiation during sustained high-frequency firing but also speed up the propagation of AP trains in PV+-BC axons by dynamically opposing the hyperpolarization produced by Na+-K+ ATPases. Since axonal AP signaling determines the timing of synaptic communication, the axon-specific expression of HCN channels represents a specialization for PV+-BCs to operate at high speed.

A Novel Glycine Receptor Variant with Startle Disease Affects Syndapin I and Glycinergic Inhibition.

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRβ subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.

Inhibitory Synapse Formation at the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential (AP) initiation in most neurons and is thus a critical site in the regulation of neuronal excitability. Normal function within the discrete AIS compartment requires intricate molecular machinery to ensure the proper concentration and organization of voltage-gated and ligand-gated ion channels; in humans, dysfunction at the AIS due to channel mutations is commonly associated with epileptic disorders. In this review, we will examine the molecular mechanisms underlying the formation of the only synapses found at the AIS: synapses containing γ-aminobutyric type A receptors (GABAARs). GABAARs are heteropentamers assembled from 19 possible subunits and are the primary mediators of fast synaptic inhibition in the brain. Although the total GABAAR population is incredibly heterogeneous, only one specific GABAAR subtype—the α2-containing receptor—is enriched at the AIS. These AIS synapses are innervated by GABAergic chandelier cells, and this inhibitory signaling is thought to contribute to the tight control of AP firing. Here, we will summarize the progress made in understanding the regulation of GABAAR synapse formation, concentrating on post-translational modifications of subunits and on interactions with intracellular proteins. We will then discuss subtype-specific synapse formation, with a focus on synapses found at the AIS, and how these synapses influence neuronal excitation.

Structure of Heteropentameric GABAA Receptors and Receptor-Anchoring Properties of Gephyrin.

γ-Aminobutyric acid type A receptors (GABAARs) mediate the majority of fast synaptic inhibition in the central nervous system (CNS). GABAARs belong to the Cys-loop superfamily of pentameric ligand-gated ion channels (pLGIC) and are assembled from 19 different subunits. As dysfunctional GABAergic neurotransmission manifests itself in neurodevelopmental disorders including epilepsy and anxiety, GABAARs are key drug targets. The majority of synaptic GABAARs are anchored at the inhibitory postsynaptic membrane by the principal scaffolding protein gephyrin, which acts as the central organizer in maintaining the architecture of the inhibitory postsynaptic density (iPSD). This interaction is mediated by the long intracellular loop located in between transmembrane helices 3 and 4 (M3–M4 loop) of the receptors and a universal receptor-binding pocket residing in the C-terminal domain of gephyrin. In 2014, the crystal structure of the β3-homopentameric GABAAR provided crucial information regarding the architecture of the receptor; however, an understanding of the structure and assembly of heteropentameric receptors at the atomic level was lacking. This review article will highlight recent advances in understanding the structure of heteropentameric synaptic GABAARs and how these structures have provided fundamental insights into the assembly of these multi-subunit receptors as well as their modulation by diverse ligands including the physiological agonist GABA. We will further discuss the role of gephyrin in the anchoring of synaptic GABAARs and glycine receptors (GlyRs), which are crucial for maintaining the architecture of the iPSD. Finally, we will also summarize how anti-malarial artemisinin drugs modulate gephyrin-mediated inhibitory neurotransmission.

Inhibition Gets a New KAR Smell.

Ionotropic and metabotropic kainate receptor signaling regulates Cl− homeostasis and GABAergic inhibition Garand D, Mahadevan V, Woodin MA. J Physiol. 2018. doi:10.1113/JP276901Potassium chloride cotransporter 2 (KCC2) plays a critical role in the regulation of chloride (Cl−) homeostasis within mature neurons. The 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. Potassium chloride cotransporter 2 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). Kainate receptors are glutamate receptors that display both ionotropic and metabotropic signaling. We show that activating KARs in the hippocampus hyperpolarizes EGABA, thus strengthening inhibition. This hyperpolarization occurs via both ionotropic and metabotropic KAR signaling in the CA3 region, whereas it is absent in the GluK1/2−/− mouse, and is independent of zinc release from mossy fiber terminals. The metabotropic signaling mechanism is dependent on KCC2, although the ionotropic signaling 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 signaling 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.