Ligand-gated Chloride Channels Research Articles

GABAA receptor signalling mechanisms revealed by structural pharmacology.

SummaryType-A γ-aminobutyric receptors (GABAARs) are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and common substances of abuse. Without reliable structural data, the mechanistic basis for pharmacological modulation of GABAARs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human α1β3γ2L GABAAR in lipid nanodiscs, bound to the channel blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA and the classical benzodiazepines alprazolam (Xanax) and diazepam (Valium), respectively. We describe the binding modes and mechanistic impacts of these ligands, the closed and desensitised states of the GABAAR gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding, and the transmembrane, pore-forming, regions. This work provides a structural framework to integrate decades of physiology and pharmacology research and a rational basis for development of novel GABAAR modulators.

International Union of Basic and Clinical Pharmacology. CVI: GABAA Receptor Subtype- and Function-selective Ligands: Key Issues in Translation to Humans.

GABAA receptors are the major inhibitory transmitter receptors in the brain. They are ligand-gated chloride channels and the site of action of benzodiazepines, barbiturates, neuroactive steroids, anesthetics, and convulsants. GABAA receptors are composed of five subunits that can belong to different subunit classes. The existence of 19 homologous subunits and their distinct regional, cellular, and subcellular distribution gives rise to a large number of GABAA receptor subtypes with distinct pharmacology, which modulate different functions of the brain. A variety of compounds have been identified that were claimed to modulate selectively individual GABAA receptor subtypes. However, many of these compounds have only incompletely been investigated or, in addition to a preferential modulation of a receptor subtype, also modulate other subtypes at similar concentrations. Although their differential efficacy at distinct receptor subtypes reduced side effects in behavioral experiments in rodents, the exact receptor subtypes mediating their behavioral effects cannot be unequivocally delineated. In addition, the discrepant in vivo effects of some of these compounds in rodents and man raised doubts on the applicability of the concept of receptor subtype selectivity as a guide for the development of clinically useful drugs. Here, we provide an up-to-date review on the currently available GABAA receptor subtype-selective ligands. We present data on their actual activity at GABAA receptor subtypes, discuss the translational aspect of subtype-selective drugs, and make proposals for the future development of ligands with better anxioselectivity in humans. Finally, we discuss possible ways to strengthen the conclusions of behavioral studies with the currently available drugs.

Okaramines and other plant fungal products as new insecticide leads.

Okaramine were indole alkaloids discovered from products of Penicillium simplicissimum AK-40 in soy bean pulp 'okara'. Okaramines exhibit insecticidal activity on a broad range of insects. Hence, more insecticide leads were explored by fermenting the other fungi with okara, resulting in the isolations of meroterpenes and cyclic peptides as well as indole alkaloids with distinct skeletons. Most okaramines activate l-glutamate-gated chloride channels found only in invertebrate nervous systems and muscle cells. Other fungal products selectively modulate other invertebrate ligand-gated chloride channels. Recently, the okaramine biosynthetic pathway has been elucidated, providing new insights in structural features important for activity. Enhanced production of okaramine in okara points to the involvement of plant mediators in the production of insect modulators by plant associated microorganisms in the rhizosphere as a novel defense strategy.

γ2 GABAAR Trafficking and the Consequences of Human Genetic Variation.

GABA type A receptors (GABAARs) mediate the majority of fast inhibitory neurotransmission in the central nervous system (CNS). Most prevalent as heteropentamers composed of two α, two β, and a γ2 subunit, these ligand-gated ionotropic chloride channels are capable of extensive genetic diversity (α1-6, β1-3, γ1-3, δ, 𝜀, 𝜃, π, ρ1-3). Part of this selective GABAAR assembly arises from the critical role for γ2 in maintaining synaptic receptor localization and function. Accordingly, mutations in this subunit account for over half of the known epilepsy-associated genetic anomalies identified in GABAARs. Fundamental structure–function studies and cellular pathology investigations have revealed dynamic GABAAR trafficking and synaptic scaffolding as critical regulators of GABAergic inhibition. Here, we introduce in vitro and in vivo findings regarding the specific role of the γ2 subunit in receptor trafficking. We then examine γ2 subunit human genetic variation and assess disease related phenotypes and the potential role of altered GABAAR trafficking. Finally, we discuss new-age imaging techniques and their potential to provide novel insight into critical regulatory mechanisms of GABAAR function.

Fluxametamide: A novel isoxazoline insecticide that acts via distinctive antagonism of insect ligand-gated chloride channels

Fluxametamide is a novel wide-spectrum insecticide that was discovered and synthesized by Nissan Chemical Industries, Ltd. To identify the mode of action of fluxametamide, we first performed [3H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB) binding assays. Fluxametamide potently inhibited the specific binding of [3H]EBOB to housefly-head membranes, suggesting that fluxametamide affects insect γ-aminobutyric acid (GABA)-gated chloride channels (GABACls). Next, the antagonism of housefly GABACls and glutamate-gated chloride channels (GluCls) was examined using the two-electrode voltage clamp (TEVC) method. Fluxametamide inhibited agonist responses in both ion channels expressed in Xenopus oocytes in the nanomolar range, indicating that this insecticide is a ligand-gated chloride channel (LGCC) antagonist. The insecticidal and LGCC antagonist potencies of fluxametamide against fipronil-susceptible and fipronil-resistant strains of small brown planthoppers and two-spotted spider mites, which are insensitive to fipronil, were evaluated. Fluxametamide exhibited similar levels of both activities in these fipronil-susceptible and fipronil-resistant arthropod pests. These data indicate that fluxametamide exerts distinctive antagonism of arthropod GABACls by binding to a site different from those for existing antagonists. In contrast to its profound actions on the arthropod LGCCs, the antagonistic activity of fluxametamide against rat GABACls and human glycine-gated chloride channels was nearly insignificant, suggesting that fluxametamide has high target-site selectivity for arthropods over mammals. Overall, fluxametamide is a new type of LGCC antagonist insecticide with excellent safety for mammals at the target-site level.

Glycine Receptor Oligomerization Characterized by Number and Brightness Analysis

Glycine receptors (GlyR) are ligand-gated chloride channels that mediate synaptic inhibition in the CNS. A loss of GlyR function by single site mutations is the major cause hyperekplexia, a rare neuromotor disorder that is characterized by exaggerated startle reflexes. The V280M mutation in the α1 subunit causes heperekplexia but, surprisingly, results in a gain of function of the receptor. The mutant receptor shows spontaneous activation and prolonged deactivation. It has been suggested that V280M might change GlyRs oligomerization and the formation of clusters. We tested this idea by using Number and Brightness (N&B) analysis to determine GlyR oligomerization. A SNAP tag was fused to the N-terminal of wt and V280M human α1 GlyR. Receptors were expressed in HEK-293 cells. Cells were incubated with the Atto-488 fluorescent probe that covalently labels SNAP tag-containing receptors. N&B analysis measures the variance in fluorescence as receptors diffuse on the surface of the basolateral cell membrane. Larger variance corresponds to larger diffusing entities - multimers of GlyRs. Our tentative stoichiometry assignment is monomer, dimer, 4-mer and 8-mer. Using outside-out patch clamp recordings of macroscopic currents, we found that the SNAP-tagged wt and V280M GlyRs had similar glycine-sensitivity and kinetics compared to the corresponding untagged receptors. Using fluorescence measurements, we found that SNAP-tagged wt receptors tended to form dimers and 4-mers as the dominant species. In the presence of 3 mM glycine ,monomers were predominant. Addition of 1 µM strychnine antagonized the effect of glycine. SNAP-tagged V280M receptors exhibit a higher level of oligomerization than wt receptors. In preliminary data from 12 cells, the 8-mer species was dominant. Further experiments are needed to determine whether changes in receptor clustering underlie the hyperekplexia phenotype of V280M α1 GlyRs.

A Single Amino Acid Substitution in the Third Transmembrane Region Has Opposite Impacts on the Selectivity of the Parasiticides Fluralaner and Ivermectin for Ligand-Gated Chloride Channels.

Fluralaner (Bravecto) is a recently marketed isoxazoline ectoparasiticide. This compound potently inhibits GABA-gated chloride channels (GABACls) and less potently glutamate-gated chloride channels (GluCls) in insects. The mechanism underlying this selectivity is unknown. Therefore, we sought to identify the amino acid residues causing the low potency of fluralaner toward GluCls. We examined the fluralaner sensitivity of mutant housefly (Musca domestica) GluCls in which amino acid residues in the transmembrane subunit interface were replaced with the positionally equivalent amino acids of Musca GABACls. Of these amino acids, substitution of an amino acid (Leu315) in the third transmembrane region (TM3) with an aromatic amino acid dramatically enhanced the potency of fluralaner in the GluCls. In stark contrast to the enhancement of fluralaner potency, this mutation eliminated the activation of currents and the potentiation but not the antagonism of glutamate responses that are otherwise all elicited by the macrolide parasiticide ivermectin (IVM). Our findings indicate that the amino acid Leu315 in Musca GluCls plays significant roles in determining the selectivity of fluralaner and IVM for these channels. Given the high sequence similarity of TM3, this may hold true more widely for the GluCls and GABACls of other insect species.

Orthosteric- versus allosteric-dependent activation of the GABAA receptor requires numerically distinct subunit level rearrangements

Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric α1β2γ2 GABAA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ1 receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA α1β2γ2 receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ1 subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits. Using these experiments, we then demonstrate that, in the pentamer, three anaesthetic-sensitive ρ1 subunits are needed to impart full efficacy to the partial GABA agonists. By contrast, five anaesthetic-sensitive subunits are required for direct activation by anaesthetics alone, and only one anaesthetic-sensitive subunit is sufficient to confer the anaesthetic-dependent potentiation to the GABA current. In conclusion, our data indicate that GABA and anaesthetics holistically activate the GABAA ρ1 receptor through distinct subunit level rearrangements and suggest that in contrast to the global impact of GABA via orthosteric sites, the force of anaesthetics through allosteric sites may not propagate to the neighbouring subunits and, thus, may have only a local and limited effect on the ρ1 GABAA receptor model system.

Effect of TPMPA (GABAC receptor antagonist) on neuronal response properties in rat barrel cortex

GABAC receptors are ligand-gated chloride channels and have important roles in some neurological functions like vision. Recent investigations demonstrated that these receptors are also expressed in the somatosensory cortex. In this study, we investigated the effect of (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) (GABAC receptor antagonist) on the properties of the neuronal response to natural stimuli (whisker deflection) in deep layers of rat barrel cortex. Twenty-eight male Wistar rats, weighing 230–260 g, were used in this study. TPMPA (100 μmol/rat) was administered intracerebroventricularly (ICV). Neuronal responses to deflection of principal (PW) and adjacent (AW) whiskers were recorded in barrel cortex using tungsten microelectrodes. A computer-controlled mechanical displacement was used to deflect whiskers individually or in combination at 30 ms inter-stimulus intervals. ON and OFF responses for PW and AW deflections were measured. A condition-test ratio (CTR) was computed to quantify neuronal responses to whisker interactions. Our data suggest that ICV administration of TPMPA increased neuronal spontaneous activity, ON and OFF responses to PW, and/or AW deflections. However, CTR for neither ON nor OFF responses changed following TPMPA administration. The results of this study demonstrated that inhibition of GABAC receptors by TPMPA can modulate neuronal response properties in rat barrel cortex.

Structure-Based Discovery of Novel Glycinergic Modulators

Marijuana's analgesic effects can be attributed to the allosteric modulation of glycine receptors (GlyRs) by Δ9-tetrahydrocannabinol (THC). GlyRs are pentameric ligand-gated chloride channels that control inhibitory neurotransmission in the brainstem and spinal cord. The glycinergic mechanism of cannabis-induced analgesia is independent of the other psychoactive effects of THC. Compounds specifically targeting the well-defined THC-binding site in GlyRs are likely to provide pain relief with fewer unwanted side effects. Here, we screened over 2 million drug-like molecules from the ZINC database on an ensemble of α3GlyR structures obtained from molecular dynamics simulations of the closed-state α3GlyR crystal structure (PDB ID: 5CFB) and a homology model derived from the open-state α1GlyR NMR structure (PDB ID: 2M6I). Computational dockings were specifically targeted to the known THC-binding site in the α3GlyR transmembrane domain. Each screened compound was ranked based on its total predicted binding affinity across the ensemble of α3GlyR structures. Top ranked compounds were selected for functional measurements in Xenopus laevis oocytes expressing human α3GlyR. Several lead candidates have been identified as strong modulators of α3GlyR, exhibiting positive and/or negative allosteric effects at micromolar concentrations. Further molecular dynamics simulations of α3GlyR in the presence of modulators revealed that in the closed-state α3GlyR, potentiating compounds showed an increased probability of contacting with residue S296 compared to inhibitors occupying the same THC-binding site. S296 on the third transmembrane helix has been previously shown to be critical for THC potentiation of GlyRs. In contrast, inhibitors showed a higher probability of close contact with residue S241 in the first transmembrane helix. Interestingly, the adjacent residue I240 has been previously reported as an inhibitory site. This study suggests that different interaction residue partners, even within the same binding site, may lead to distinctly different allosteric modulations in α3GlyR. Research supported by NIH grants.

Evolution, Expression, and Function of Nonneuronal Ligand-Gated Chloride Channels in Drosophila melanogaster.

Ligand-gated chloride channels have established roles in inhibitory neurotransmission in the nervous systems of vertebrates and invertebrates. Paradoxically, expression databases in Drosophila melanogaster have revealed that three uncharacterized ligand-gated chloride channel subunits, CG7589, CG6927, and CG11340, are highly expressed in nonneuronal tissues. Furthermore, subunit copy number varies between insects, with some orders containing one ortholog, whereas other lineages exhibit copy number increases. Here, we show that the Dipteran lineage has undergone two gene duplications followed by expression-based functional differentiation. We used promoter-GFP expression analysis, RNA-sequencing, and in situ hybridization to examine cell type and tissue-specific localization of the three D. melanogaster subunits. CG6927 is expressed in the nurse cells of the ovaries. CG7589 is expressed in multiple tissues including the salivary gland, ejaculatory duct, malpighian tubules, and early midgut. CG11340 is found in malpighian tubules and the copper cell region of the midgut. Overexpression of CG11340 increased sensitivity to dietary copper, and RNAi and ends-out knockout of CG11340 resulted in copper tolerance, providing evidence for a specific nonneuronal role for this subunit in D. melanogaster. Ligand-gated chloride channels are important insecticide targets and here we highlight copy number and functional divergence in insect lineages, raising the potential that order-specific receptors could be isolated within an effective class of insecticide targets.

ChemInform Abstract: Isoxazolines: A Novel Chemotype Highly Effective on Ectoparasites

AbstractReview: 60 refs.

Isoxazolines: A Novel Chemotype Highly Effective on Ectoparasites.

Efficient control of arthropod ectoparasite infestations has a long-standing history in the agriculture and veterinary sectors, aiming to decrease the parasite burden of affected crops and animals. Ligand-gated chloride channels (LGCCs) modulated by γ-aminobutyric acid (GABA) and glutamate have been identified as suitable molecular targets, and several classes of potent parasiticides have been devised. Due to the increase in cross-resistance and decreased development of new chemical entities, an urgent need for new parasiticides or prevention schemes has emerged. In the last decade, an innovative isoxazoline chemotype appears to offer promise for inhibiting LGCCs with a new mode of action and distinct binding site from that of historical agents. Considerable efforts have focused on optimizing the antiparasitic activity of isoxazolines and may provide the potential for future human use.

Genetic and functional analyses demonstrate a role for abnormal glycinergic signaling in autism.

Autism spectrum disorder (ASD) is a common neurodevelopmental condition characterized by marked genetic heterogeneity. Recent studies of rare structural and sequence variants have identified hundreds of loci involved in ASD, but our knowledge of the overall genetic architecture and the underlying pathophysiological mechanisms remains incomplete. Glycine receptors (GlyRs) are ligand-gated chloride channels that mediate inhibitory neurotransmission in the adult nervous system but exert an excitatory action in immature neurons. GlyRs containing the α2 subunit are highly expressed in the embryonic brain, where they promote cortical interneuron migration and the generation of excitatory projection neurons. We previously identified a rare microdeletion of the X-linked gene GLRA2, encoding the GlyR α2 subunit, in a boy with autism. The microdeletion removes the terminal exons of the gene (GLRA2(Δex8-9)). Here, we sequenced 400 males with ASD and identified one de novo missense mutation, p.R153Q, absent from controls. In vitro functional analysis demonstrated that the GLRA2(Δex8)(-)(9) protein failed to localize to the cell membrane, while the R153Q mutation impaired surface expression and markedly reduced sensitivity to glycine. Very recently, an additional de novo missense mutation (p.N136S) was reported in a boy with ASD, and we show that this mutation also reduced cell-surface expression and glycine sensitivity. Targeted glra2 knockdown in zebrafish induced severe axon-branching defects, rescued by injection of wild type but not GLRA2(Δex8-9) or R153Q transcripts, providing further evidence for their loss-of-function effect. Glra2 knockout mice exhibited deficits in object recognition memory and impaired long-term potentiation in the prefrontal cortex. Taken together, these results implicate GLRA2 in non-syndromic ASD, unveil a novel role for GLRA2 in synaptic plasticity and learning and memory, and link altered glycinergic signaling to social and cognitive impairments.

Abamectin induces rapid and reversible hypoactivity within early zebrafish embryos

During early zebrafish embryogenesis, spontaneous tail contractions represent the first sign of locomotion and result from innervation of primary motoneuron axons to target axial muscles. Based on a high-content screen, we previously demonstrated that exposure of zebrafish embryos to abamectin – an avermectin insecticide – from 5–25hours post-fertilization (hpf) abolished spontaneous activity in the absence of effects on survival and gross morphology. Therefore, the objective of this study was to begin investigating the mechanism of abamectin-induced hypoactivity in zebrafish. Similar to 384-well plates, static exposure of embryos to abamectin from 5–25hpf in glass beakers resulted in elimination of activity at low micromolar concentrations. However, abamectin did not affect neurite outgrowth from spinal motoneurons and, compared with exposure from 5–25hpf, embryos were equally susceptible to abamectin-induced hypoactivity when exposures were initiated at 10 and 23hpf. Moreover, immersion of abamectin-exposed embryos in clean water resulted in complete recovery of spontaneous activity relative to vehicle controls, suggesting that abamectin reversibly activated ligand-gated chloride channels and inhibited neurotransmission. To test this hypothesis, we pretreated embryos to vehicle or non-toxic concentrations of fipronil or endosulfan – two insecticides that antagonize the γ-aminobutyric acid (GABA) receptor – from 5–23hpf, and then exposed embryos to vehicle or abamectin from 23–25hpf. Interestingly, activity levels within abamectin-exposed embryos pretreated with either antagonist were similar to embryos exposed to vehicle alone. Using quantitative PCR and phylogenetic analyses, we then confirmed the presence of GABA receptor α1 and β2 subunits at 5, 10, and 23hpf, and demonstrated that zebrafish GABA receptor subunits are homologous to mammalian GABA receptor subunits. Overall, our data collectively suggest that abamectin induces rapid and reversible hypoactivity within early zebrafish embryos, an effect that may be mediated through the GABA receptor.

The Kinetic Properties of the Human Glycine Receptor in Response to Different Agonists

Glycine receptors are ligand-gated chloride channels that mediate much of the fast synaptic inhibition in the spinal cord and brainstem. They can exist as homomers (formed by five α1 subunits), or as heteromers (formed by 3 α1 subunits and 2 β subunits). One of our current lines of work is to understand the effect on glycine receptors of a range of agonist molecules with structures systematically changed from that of glycine. In this study, we selected two agonists, β-alanine and D-alanine, for detailed single channel analysis. By fitting kinetic schemes to cell-attached data we were able to get insight into the binding and gating of the glycine receptor in response to the different agonists. We also used concentration jump experiments in the outside-out configuration to validate the fitted data. Molecular dynamics simulations also allowed us to see how the agonists docked into the binding site.While binding and gating rate constants remained relatively similar between all agonists, the ability of the agonists to stabilise the pre-open flipped confirmation varied in a manner similar to that reported by Lape et al. (Nature, 2008. 454 722-729). The less effective agonist D-alanine proved also to have a lower binding affinity than the other two agonists. Molecular docking studies were able to confirm that both D-alanine and β-alanine bind in a similar fashion to glycine. However, β-alanine shows a lot more mobility in the binding site, and this perhaps accounts for the large amount of heterogeneity in single channel recordings with this agonist.

Glycine Protects Hepatocytes through a Chloride Independent Mechanism

Cytoprotection by the amino acid glycine was first described more than two decades ago. Accumulated evidence shows that glycine protects against hypoxia, ischemia-reperfusion and several toxins in hepatocytes, renal tubular cells and other cell types. The exact mechanism of the protection is still under investigation and multiple models have been proposed. One working model is that the protection exerted by glycine is mediated by inhibition of ligand-gated chloride channels. In our study, we used a genetically encoded chloride biosensor to test this working model. Isolated rat hepatocytes infected with adenovirus for expression of the chloride biosensor were treated with the mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), in the presence and absence of 3 mM glycine. With and without glycine, intracellular chloride concentration increased upon addition of CCCP. Without glycine, CCCP led to swelling, membrane bursting and death of hepatocytes in Krebs-Ringer-Hepes buffer. By contrast in the presence of glycine, cell death was markedly delayed. Moreover instead of swelling, hepatocytes underwent cell shrinkage after CCCP. These findings suggest that glycine alters cell volume regulation after CCCP and prevents cellular swelling leading to plasma membrane rupture and consequent loss of cell viability. DK073336, DK037034 and 14.Z50.31.0028.

Syndrome of severe pain associated with a continuous bumetanide infusion

Syndrome of severe pain associated with a continuous bumetanide infusion

The role of Rdl in resistance to phenylpyrazoles in Drosophila melanogaster

Extensive use of older generation insecticides may result in pre-existing cross-resistance to new chemical classes acting at the same target site. Phenylpyrazole insecticides block inhibitory neurotransmission in insects via their action on ligand-gated chloride channels (LGCCs). Phenylpyrazoles are broad-spectrum insecticides widely used in agriculture and domestic pest control. So far, all identified cases of target site resistance to phenylpyrazoles are based on mutations in the Rdl (Resistance to dieldrin) LGCC subunit, the major target site for cyclodiene insecticides. We examined the role that mutations in Rdl have on phenylpyrazole resistance in Drosophila melanogaster, exploring naturally occurring variation, and generating predicted resistance mutations by mutagenesis. Natural variation at the Rdl locus in inbred strains of D. melanogaster included gene duplication, and a line containing two Rdl mutations found in a highly resistant line of Drosophila simulans. These mutations had a moderate impact on survival following exposure to two phenylpyrazoles, fipronil and pyriprole. Homology modelling suggested that the Rdl chloride channel pore contains key residues for binding fipronil and pyriprole. Mutagenesis of these sites and assessment of resistance in vivo in transgenic lines showed that amino acid identity at the Ala301 site influenced resistance levels, with glycine showing greater survival than serine replacement. We confirm that point mutations at the Rdl 301 site provide moderate resistance to phenylpyrazoles in D. melanogaster. We also emphasize the beneficial aspects of testing predicted mutations in a whole organism to validate a candidate gene approach.

Modeling the effects of neuronal morphology on dendritic chloride diffusion and GABAergic inhibition

Poster presentation at the Twenty Third Annual Computational Neuroscience Meeting: CNS*2014 Quebec City, Canada. 26-31 July 2014. Gamma-aminobutyric acid receptors (GABAARs) are ligand-gated chloride (Cl−) channels which mediate the majority of inhibitory neurotransmission in the CNS. Spatiotemporal changes of intracellular Cl− concentration alter the concentration gradient for Cl− across the neuronal membrane and thus affect the current flow through GABAARs and the efficacy of GABAergic inhibition. However, the impact of complex neuronal morphology on Cl− diffusion and the redistribution of intracellular Cl− is not well understood. Recently, computational models for Cl− diffusion and GABAAR-mediated inhibition in realistic neuronal morphologies became available [1-3]. Here we have used computational models of morphologically complex dendrites to test the effects of spines on Cl− diffusion. In all dendritic morphologies tested, spines slowed down longitudinal Cl− diffusion along dendrites and decreased the amount and spatial spread of synaptically evoked Cl− changes. Spine densities of 2-10 spines/µm decreased the longitudinal diffusion coefficient of Cl− to 80-30% of its value in smooth dendrites, respectively. These results suggest that spines are able to limit short-term ionic plasticity [4] at dendritic GABAergic synapses.