RESUMEN
Many therapeutic antibodies deplete target cells and elicit immunotherapy by engaging activating Fc gamma receptors (FcγRs) on host effector cells. These antibodies are negatively regulated by the inhibitory FcγRIIB (CD32B). Dogma suggests inhibition is mediated through the FcγRIIB immunoreceptor tyrosine-based inhibition motif (ITIM), negatively regulating immunoreceptor tyrosine-based activation motif (ITAM)-mediated signaling from activating FcγR. To assess this, we generated experimental models expressing human (h)FcγRIIB on targets or effectors, lacking or retaining ITIM signaling capacity. We demonstrate that signaling through the hFcγRIIB ITIM is dispensable for impairing monoclonal antibody (mAb)-mediated depletion of normal and malignant murine target cells through three therapeutically relevant surface receptors (CD20, CD25, and OX40) affecting immunotherapy. We demonstrate that hFcγRIIB competition with activating FcγRs for antibody Fc, rather than ITIM signaling, is sufficient to impair activating FcγR engagement, inhibiting effector function and immunotherapy.
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Anticuerpos Monoclonales , Receptores de IgG/inmunología , Animales , Humanos , Inmunoterapia , Ratones , Receptores de IgG/metabolismo , Transducción de SeñalRESUMEN
The 3,9-diazaspiro[5.5]undecane-based compounds 2027 and 018 have previously been reported to be potent competitive γ-aminobutyric acid type A receptor (GABAAR) antagonists showing low cellular membrane permeability. Given the emerging peripheral application of GABAAR ligands, we hypothesize 2027 analogs as promising lead structures for peripheral GABAAR inhibition. We herein report a study on the structural determinants of 2027 in order to suggest a potential binding mode as a basis for rational design. The study identified the importance of the spirocyclic benzamide, compensating for the conventional acidic moiety, for GABAAR ligands. The structurally simplified m-methylphenyl analog 1e displayed binding affinity in the high-nanomolar range (Ki = 180 nM) and was superior to 2027 and 018 regarding selectivity for the extrasynaptic α4ßδ subtype versus the α1- and α2- containing subtypes. Importantly, 1e was shown to efficiently rescue inhibition of T cell proliferation, providing a platform to explore the immunomodulatory potential for this class of compounds.
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Adyuvantes Inmunológicos/farmacología , Alcanos/farmacología , Antagonistas del GABA/farmacología , Receptores de GABA-A/efectos de los fármacos , Adyuvantes Inmunológicos/química , Alcanos/química , Proliferación Celular/efectos de los fármacos , Antagonistas del GABA/química , Humanos , Relación Estructura-Actividad , Linfocitos T/citología , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: The 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. METHODS: Mouse 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. RESULTS: Positive 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. CONCLUSIONS: We conclude that activation of GABA(A)Rs provides immunosuppression by inhibiting T cell proliferation.
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Benzodiazepinas/farmacología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular/efectos de los fármacos , Pregnanolona/farmacología , Receptores de GABA-A/metabolismo , Animales , Humanos , Ratones , Receptores de GABA/metabolismoRESUMEN
Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Na v1.8 Cre and then crossed to CGRP CreER (Calca), Th CreERT2, Tmem45b Cre, Tmem233 Cre, Ntng1 Cre and TrkB CreER (Ntrk2) lines. Pain behavioural assays included Hargreaves', hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Na v1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders.
RESUMEN
Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A unique approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. Bacterial artificial chromosome (BAC) transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human pain-insensitive phenotype are therefore potential novel targets for the development of new analgesic drugs.awx326media15680039660001.
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Insensibilidad Congénita al Dolor/genética , Umbral del Dolor/fisiología , Dolor/fisiopatología , Mutación Puntual/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Adolescente , Adulto , Anciano , Animales , Calcio/metabolismo , Capsaicina/efectos adversos , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Humanos , Hiperalgesia/patología , Hiperalgesia/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Dolor/inducido químicamente , Insensibilidad Congénita al Dolor/patología , Insensibilidad Congénita al Dolor/fisiopatología , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Piel/patología , Adulto JovenRESUMEN
Loss-of-function mutations in the SCN9A gene encoding voltage-gated sodium channel Nav1.7 cause congenital insensitivity to pain in humans and mice. Surprisingly, many potent selective antagonists of Nav1.7 are weak analgesics. We investigated whether Nav1.7, as well as contributing to electrical signalling, may have additional functions. Here we report that Nav1.7 deletion has profound effects on gene expression, leading to an upregulation of enkephalin precursor Penk mRNA and met-enkephalin protein in sensory neurons. In contrast, Nav1.8-null mutant sensory neurons show no upregulated Penk mRNA expression. Application of the opioid antagonist naloxone potentiates noxious peripheral input into the spinal cord and dramatically reduces analgesia in both female and male Nav1.7-null mutant mice, as well as in a human Nav1.7-null mutant. These data suggest that Nav1.7 channel blockers alone may not replicate the analgesic phenotype of null mutant humans and mice, but may be potentiated with exogenous opioids.
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Encefalinas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Insensibilidad Congénita al Dolor/metabolismo , Adulto , Animales , Encefalinas/genética , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.7/genética , Insensibilidad Congénita al Dolor/genética , Insensibilidad Congénita al Dolor/fisiopatología , Sensación , Células Receptoras Sensoriales/metabolismoRESUMEN
BACKGROUND: Tamoxifen (TAM) is an important cancer therapeutic and an experimental tool for effecting genetic recombination using the inducible Cre-Lox technique. Despite its widespread use in the clinic and laboratory, we know little about its effects on the nervous system. This is of significant concern because TAM, via unknown mechanisms, induces cognitive impairment in humans. A hallmark of cellular stress is induction of Activating Transcription Factor 3 (Atf3), and so to determine whether TAM induces cellular stress in the adult nervous system, we generated a knock-in mouse in which Atf3 promoter activity drives transcription of TAM-dependent Cre recombinase (Cre-ERT2); when crossed with tdtomato reporter mice, Atf3 induction results in robust and permanent genetic labeling of cells in which it is up-regulated even transiently. RESULTS: We found that granular neurons of the olfactory bulb and dentate gyrus, vascular cells and ependymal cells throughout the brain, and peripheral sensory neurons expressed tdtomato in response to TAM treatment. We also show that TAM induced Atf3 up-regulation through inhibition of cholesterol epoxide hydrolase (ChEH): reporter expression was mitigated by delivery in vitamin E-rich wheat germ oil (vitamin E depletes ChEH substrates), and was partially mimicked by a ChEH-specific inhibitor. CONCLUSIONS: This work demonstrates that TAM stresses cells of the adult central and peripheral nervous systems and highlights concerns about clinical and experimental use of TAM. We propose TAM administration in vitamin E-rich vehicles such as wheat germ oil as a simple remedy.
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Colesterol/metabolismo , Sistema Nervioso/citología , Neuronas/fisiología , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Regulación hacia Arriba/efectos de los fármacos , Factor de Transcripción Activador 3/genética , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Relación Dosis-Respuesta a Droga , Epóxido Hidrolasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Aceites de Plantas/farmacología , Regiones Promotoras Genéticas , Vitamina E/farmacologíaRESUMEN
Transcriptional alterations are characteristic of persistent pain states, but the key regulators remain elusive. HDAC4 is a transcriptional corepressor that has been linked to synaptic plasticity and neuronal excitability, mechanisms that may be involved in peripheral and central sensitization. Using a conditional knockout (cKO) strategy in mice, we sought to determine whether the loss of HDAC4 would have implications for sensory neuron transcription and nociception. HDAC4 was found to be largely unnecessary for transcriptional regulation of naïve sensory neurons but was essential for appropriate transcriptional responses after injury, with Calca and Trpv1 expression consistently down-regulated in HDAC4 cKO compared to levels in the littermate controls (0.2-0.44-fold change, n = 4 in 2 separate experiments). This down-regulation corresponded to reduced sensitivity to 100 nM capsaicin in vitro (IC50 = 230 ± 20 nM, 76 ± 4.4% wild-type capsaicin responders vs. 56.9 ± 4.7% HDAC4 cKO responders) and to reduced thermal hypersensitivity in the complete Freund's adjuvant (CFA) model of inflammatory pain (1.3-1.4-fold improvement over wild-type controls; n = 5-12, in 2 separate experiments). These data indicate that HDAC4 is a novel inflammatory pain mediator and may be a good therapeutic target, capable of orchestrating the regulation of multiple downstream effectors.
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Histona Desacetilasas/metabolismo , Hiperalgesia/metabolismo , Hipersensibilidad/metabolismo , Inflamación/metabolismo , Animales , Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina , Capsaicina/farmacología , Regulación hacia Abajo/efectos de los fármacos , Adyuvante de Freund/farmacología , Hiperalgesia/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nocicepción/efectos de los fármacos , Nocicepción/fisiología , Dolor/tratamiento farmacológico , Dolor/metabolismo , Precursores de Proteínas/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/metabolismo , Canales Catiónicos TRPV/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiologíaRESUMEN
Nav1.7, a peripheral neuron voltage-gated sodium channel, is essential for pain and olfaction in mice and humans. We examined the role of Nav1.7 as well as Nav1.3, Nav1.8, and Nav1.9 in different mouse models of chronic pain. Constriction-injury-dependent neuropathic pain is abolished when Nav1.7 is deleted in sensory neurons, unlike nerve-transection-related pain, which requires the deletion of Nav1.7 in sensory and sympathetic neurons for pain relief. Sympathetic sprouting that develops in parallel with nerve-transection pain depends on the presence of Nav1.7 in sympathetic neurons. Mechanical and cold allodynia required distinct sets of neurons and different repertoires of sodium channels depending on the nerve injury model. Surprisingly, pain induced by the chemotherapeutic agent oxaliplatin and cancer-induced bone pain do not require the presence of Nav1.7 sodium channels or Nav1.8-positive nociceptors. Thus, similar pain phenotypes arise through distinct cellular and molecular mechanisms. Therefore, rational analgesic drug therapy requires patient stratification in terms of mechanisms and not just phenotype.
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Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Neuralgia/metabolismo , Dolor Nociceptivo/metabolismo , Nociceptores/metabolismo , Animales , Hiperalgesia/metabolismo , Ratones , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.8/genética , Canal de Sodio Activado por Voltaje NAV1.8/metabolismo , Neuralgia/genética , Nocicepción , Dolor Nociceptivo/genética , Nociceptores/fisiologíaRESUMEN
BACKGROUND: Tissue-specific gene deletion has proved informative in the analysis of pain pathways. Advillin has been shown to be a pan-neuronal marker of spinal and cranial sensory ganglia. We generated BAC transgenic mice using the Advillin promoter to drive a tamoxifen-inducible CreERT2 recombinase construct in order to be able to delete genes in adult animals. We used a floxed stop ROSA26LacZ reporter mouse to examine functional Cre expression, and analysed the behaviour of mice expressing Cre recombinase. RESULTS: We used recombineering to introduce a CreERT2 cassette in place of exon 2 of the Advillin gene into a BAC clone (RPCI23-424F19) containing the 5' region of the Advillin gene. Transgenic mice were generated using pronuclear injection. The resulting AvCreERT2 transgenic mice showed a highly specific expression pattern of Cre activity after tamoxifen induction. Recombinase activity was confined to sensory neurons and no expression was found in other organs. Less than 1% of neurons showed Cre expression in the absence of tamoxifen treatment. Five-day intraperitoneal treatment with tamoxifen (2 mg per day) induced Cre recombination events in ≈90% of neurons in dorsal root and cranial ganglia. Cell counts of dorsal root ganglia (DRG) from transgenic animals with or without tamoxifen treatment showed no neuronal cell loss. Sensory neurons in culture showed ≈70% induction after 3 days treatment with tamoxifen. Behavioural tests showed no differences between wildtype, AvCreERT2 and tamoxifen-treated animals in terms of motor function, responses to light touch and noxious pressure, thermal thresholds as well as responses to inflammatory agents. CONCLUSIONS: Our results suggest that the inducible pan-DRG AvCreERT2 deleter mouse strain is a useful tool for studying the role of individual genes in adult sensory neuron function. The pain phenotype of the Cre-induced animal is normal; therefore any alterations in pain processing can be unambiguously attributed to loss of the targeted gene.
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Ganglios Sensoriales/efectos de los fármacos , Eliminación de Gen , Proteínas de Microfilamentos/genética , Receptores de Estrógenos/genética , Proteínas Recombinantes de Fusión/genética , Tamoxifeno/farmacología , Animales , Células Cultivadas , Ganglios Sensoriales/metabolismo , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Recombinación Genética , Células Receptoras Sensoriales/metabolismoRESUMEN
GABAA receptors mediate the majority of fast synaptic inhibition in the brain. The accumulation of these ligand-gated ion channels at synaptic sites is a prerequisite for neuronal inhibition, but the molecular mechanisms underlying this phenomenon remain obscure. To further understand these processes, we have examined the cellular origins of synaptic GABAA receptors. To do so, we have created fluorescent GABAA receptors that are capable of binding -bungarotoxin (Bgt), facilitating the visualization of receptor endocytosis, exocytosis and delivery to synaptic sites. Imaging with Bgt in hippocampal neurons revealed that GABAA receptor endocytosis occurred exclusively at extrasynaptic sites, consistent with the preferential colocalization of extrasynaptic receptors with the AP2 adaptin. Receptor insertion into the plasma membrane was also predominantly extrasynaptic, and pulse-chase analysis revealed that these newly inserted receptors were then able to access directly synaptic sites. Therefore, our results demonstrate that synaptic GABAA receptors are directly recruited from their extrasynaptic counterparts. Moreover, they illustrate a dynamic mechanism for neurons to modulate GABAA receptor number at inhibitory synapses by controlling the stability of extrasynaptic receptors.
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Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Animales , Transporte Biológico Activo , Bungarotoxinas , Línea Celular , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Endocitosis , Colorantes Fluorescentes , Hipocampo/metabolismo , Humanos , Cinética , Neuronas/metabolismo , Receptores de GABA-A/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
The efficacy of fast synaptic inhibition is critically dependent on the accumulation of GABAA receptors at inhibitory synapses, a process that remains poorly understood. Here, we examined the dynamics of cell surface GABAA receptors using receptor subunits modified with N-terminal extracellular ecliptic pHluorin reporters. In hippocampal neurons, GABAA receptors incorporating pHluorin-tagged subunits were found to be clustered at synaptic sites and also expressed as diffuse extrasynaptic staining. By combining FRAP (fluorescence recovery after photobleaching) measurements with live imaging of FM4-64-labeled active presynaptic terminals, it was evident that clustered synaptic receptors exhibit significantly lower rates of mobility at the cell surface compared with their extrasynaptic counterparts. To examine the basis of this confinement, we used RNAi to inhibit the expression of gephyrin, a protein shown to regulate the accumulation of GABAA receptors at synaptic sites. However, whether gephyrin acts to control the actual formation of receptor clusters, their stability, or is simply a global regulator of receptor cell surface number remains unknown. Inhibiting gephyrin expression did not modify the total number of GABAA receptors expressed on the neuronal cell surface but significantly decreased the number of receptor clusters. Live imaging revealed that clusters that formed in the absence of gephyrin were significantly more mobile compared with those in control neurons. Together, our results demonstrate that synaptic GABAA receptors have lower levels of lateral mobility compared with their extrasynaptic counterparts, and suggest a specific role for gephyrin in reducing the diffusion of GABAA receptors, facilitating their accumulation at inhibitory synapses.
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Proteínas Portadoras/fisiología , Proteínas de la Membrana/fisiología , Inhibición Neural/fisiología , Dinámicas no Lineales , Receptores de GABA-A/metabolismo , Sinapsis/fisiología , Animales , Biotinilación/métodos , Western Blotting/métodos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Clonación Molecular/métodos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Humanos , Inmunohistoquímica/métodos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Proteínas de la Membrana/genética , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fotoblanqueo , Terminales Presinápticos/metabolismo , Subunidades de Proteína/metabolismo , Compuestos de Piridinio/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Interferencia de ARN/fisiología , Ratas , Receptores AMPA/metabolismo , Receptores de GABA-A/genética , Serina-Treonina Quinasas TOR , Transfección/métodos , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
The efficacy of synaptic inhibition depends on the number of gamma-aminobutyric acid type A receptors (GABA(A)Rs) expressed on the cell surface of neurons. The clathrin adaptor protein 2 (AP2) complex is a critical regulator of GABA(A)R endocytosis and, hence, surface receptor number. Here, we identify a previously uncharacterized atypical AP2 binding motif conserved within the intracellular domains of all GABA(A)R beta subunit isoforms. This AP2 binding motif (KTHLRRRSSQLK in the beta3 subunit) incorporates the major sites of serine phosphorylation within receptor beta subunits, and phosphorylation within this site inhibits AP2 binding. Furthermore, by using surface plasmon resonance, we establish that a peptide (pepbeta3) corresponding to the AP2 binding motif in the GABA(A)R beta3 subunit binds to AP2 with high affinity only when dephosphorylated. Moreover, the pepbeta3 peptide, but not its phosphorylated equivalent (pepbeta3-phos), enhanced the amplitude of miniature inhibitory synaptic current and whole cell GABA(A)R current. These effects of pepbeta3 on GABA(A)R current were occluded by inhibitors of dynamin-dependent endocytosis supporting an action of pepbeta3 on GABA(A)R endocytosis. Therefore phospho-dependent regulation of AP2 binding to GABA(A)Rs provides a mechanism to specify receptor cell surface number and the efficacy of inhibitory synaptic transmission.