RESUMEN
GABAB receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABAB receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K+-channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABAB receptors also associate with hetero-oligomers of KCTD subunits. Coimmunoprecipitation experiments indicate that two-thirds of the KCTD16 proteins in the hippocampus of adult mice associate with KCTD12. We show that the KCTD proteins hetero-oligomerize through self-interacting T1 and H1 homology domains. Bioluminescence resonance energy transfer measurements in live cells reveal that KCTD12/KCTD16 hetero-oligomers associate with both the receptor and the G-protein. Electrophysiological experiments demonstrate that KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties on G-protein-activated Kir3 currents. During prolonged receptor activation (one min) KCTD12/KCTD16 hetero-oligomers produce moderately desensitizing fast deactivating K+ currents, whereas KCTD12 and KCTD16 homo-oligomers produce strongly desensitizing fast deactivating currents and nondesensitizing slowly deactivating currents, respectively. During short activation (2 s) KCTD12/KCTD16 hetero-oligomers produce nondesensitizing slowly deactivating currents. Electrophysiological recordings from hippocampal neurons of KCTD knock-out mice are consistent with these findings and indicate that KCTD12/KCTD16 hetero-oligomers increase the duration of slow IPSCs. In summary, our data demonstrate that simultaneous assembly of distinct KCTDs at the receptor increases the molecular and functional repertoire of native GABAB receptors and modulates physiologically induced K+ current responses in the hippocampus. SIGNIFICANCE STATEMENT: The KCTD proteins 8, 12, and 16 are auxiliary subunits of GABAB receptors that differentially regulate G-protein signaling of the receptor. The KCTD proteins are generally assumed to function as homo-oligomers. Here we show that the KCTD proteins also assemble hetero-oligomers in all possible dual combinations. Experiments in live cells demonstrate that KCTD hetero-oligomers form at least tetramers and that these tetramers directly interact with the receptor and the G-protein. KCTD12/KCTD16 hetero-oligomers impart unique kinetic properties to GABAB receptor-induced Kir3 currents in heterologous cells. KCTD12/KCTD16 hetero-oligomers are abundant in the hippocampus, where they prolong the duration of slow IPSCs in pyramidal cells. Our data therefore support that KCTD hetero-oligomers modulate physiologically induced K+ current responses in the brain.
Asunto(s)
Canales de Potasio/genética , Canales de Potasio/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Animales , Química Encefálica/genética , Células CHO , Cricetinae , Cricetulus , Fenómenos Electrofisiológicos/genética , Potenciales Postsinápticos Excitadores/genética , Femenino , Cinética , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Receptores KIR/metabolismoRESUMEN
GABA(B) receptors are the G-protein-coupled receptors for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. They are expressed in almost all neurons of the brain, where they regulate synaptic transmission and signal propagation by controlling the activity of voltage-gated calcium (Ca(v)) and inward-rectifier potassium (K(ir)) channels. Molecular cloning revealed that functional GABA(B) receptors are formed by the heteromeric assembly of GABA(B1) with GABA(B2) subunits. However, cloned GABA(B(1,2)) receptors failed to reproduce the functional diversity observed with native GABA(B) receptors. Here we show by functional proteomics that GABA(B) receptors in the brain are high-molecular-mass complexes of GABA(B1), GABA(B2) and members of a subfamily of the KCTD (potassium channel tetramerization domain-containing) proteins. KCTD proteins 8, 12, 12b and 16 show distinct expression profiles in the brain and associate tightly with the carboxy terminus of GABA(B2) as tetramers. This co-assembly changes the properties of the GABA(B(1,2)) core receptor: the KCTD proteins increase agonist potency and markedly alter the G-protein signalling of the receptors by accelerating onset and promoting desensitization in a KCTD-subtype-specific manner. Taken together, our results establish the KCTD proteins as auxiliary subunits of GABA(B) receptors that determine the pharmacology and kinetics of the receptor response.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Conductividad Eléctrica , Agonistas de Receptores GABA-B , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Cinética , Ratones , Neuronas/metabolismo , Oocitos/metabolismo , Potasio/metabolismo , Canales de Potasio/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Transducción de Señal , XenopusRESUMEN
GABA(B) receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA(B) receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA(B1a), GABA(B1b), and GABA(B2) form fully functional heteromeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA(B) receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA(B) receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA(B) receptor-mediated K(+) current response. In summary, our experiments support that the up-regulation of functional GABA(B) receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.
Asunto(s)
Hipocampo/metabolismo , Neuronas/metabolismo , Canales de Potasio/metabolismo , Potasio/metabolismo , Multimerización de Proteína/fisiología , Receptores de GABA-B/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Hipocampo/citología , Ratones , Ratones Noqueados , Neuronas/citología , Canales de Potasio/genética , Receptores de GABA-B/genéticaRESUMEN
Functional asymmetry of G-protein-coupled receptor (GPCR) dimers has been reported for an increasing number of cases, but the molecular architecture of signalling units associated to these dimers remains unclear. Here, we characterized the molecular complex of the melatonin MT1 receptor, which directly and constitutively couples to G(i) proteins and the regulator of G-protein signalling (RGS) 20. The molecular organization of the ternary MT1/G(i)/RGS20 complex was monitored in its basal and activated state by bioluminescence resonance energy transfer between probes inserted at multiple sites of the complex. On the basis of the reported crystal structures of G(i) and the RGS domain, we propose a model wherein one G(i) and one RGS20 protein bind to separate protomers of MT1 dimers in a pre-associated complex that rearranges upon agonist activation. This model was further validated with MT1/MT2 heterodimers. Collectively, our data extend the concept of asymmetry within GPCR dimers, reinforce the notion of receptor specificity for RGS proteins and highlight the advantage of GPCRs organized as dimers in which each protomer fulfils its specific task by binding to different GPCR-interacting proteins.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptor de Melatonina MT1/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Electrofisiología , Transferencia de Energía , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Inmunoprecipitación , Riñón/citología , Riñón/metabolismo , Melatonina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas RGS , Receptor de Melatonina MT1/química , Receptor de Melatonina MT1/genética , Transducción de SeñalRESUMEN
GABAB receptors (GBRs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GBRs regulate fast synaptic transmission by gating Ca2+ and K+ channels via the Gßγ subunits of the activated G protein. It has been demonstrated that auxiliary GBR subunits, the KCTD proteins, shorten onset and rise time and increase desensitization of receptor-induced K+ currents. KCTD proteins increase desensitization of K+ currents by scavenging Gßγ from the channel, yet the mechanism responsible for the rapid activation of K+ currents has remained elusive. In this study, we demonstrate that KCTD proteins preassemble Gßγ at GBRs. The preassembly obviates the need for diffusion-limited G protein recruitment to the receptor, thereby accelerating G protein activation and, as a result, K+ channel activation. Preassembly of Gßγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gß subunits. The binding site is shared between Gß1 and Gß2, limiting the interaction of KCTD proteins to these particular Gß isoforms. Substituting residues in the KCTD binding site of Gß1 with those from Gß3 hinders the preassembly of Gßγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gß interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.
RESUMEN
The properties of glycine receptors (GlyRs) depend upon their subunit composition. While the prevalent adult forms of GlyRs are heteromers, previous reports suggested functional α homomeric receptors in mature nervous tissues. Here we show two functionally different GlyRs populations in the rat medial nucleus of trapezoid body (MNTB). Postsynaptic receptors formed α1/ß-containing clusters on somatodendritic domains of MNTB principal neurons, colocalizing with glycinergic nerve endings to mediate fast, phasic IPSCs. In contrast, presynaptic receptors on glutamatergic calyx of Held terminals were composed of dispersed, homomeric α1 receptors. Interestingly, the parent cell bodies of the calyces of Held, the globular bushy cells of the cochlear nucleus, expressed somatodendritic receptors (α1/ß heteromers) and showed similar clustering and pharmacological profile as GlyRs on MNTB principal cells. These results suggest that specific targeting of GlyR ß-subunit produces segregation of GlyR subtypes involved in two different mechanisms of modulation of synaptic strength.
Asunto(s)
Vías Auditivas/metabolismo , Receptores de Glicina/metabolismo , Sinapsis/metabolismo , Animales , Espinas Dendríticas/fisiología , Estimulación Eléctrica , Fenómenos Electrofisiológicos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Glicina/fisiología , Glicinérgicos/farmacología , Inmunohistoquímica , Cinética , Microscopía Inmunoelectrónica , Terminaciones Nerviosas/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , Receptores de Glicina/efectos de los fármacos , Receptores Presinapticos/metabolismoRESUMEN
The calyx of Held synapse of the medial nucleus of the trapezoid body is a giant axosomatic synapse in the auditory brainstem, which acts as a relay synapse showing little dependence of its synaptic strength on firing frequency. The main mechanism that is responsible for its resistance to synaptic depression is its large number of release sites with low release probability. Here, we investigated the contribution of presynaptic GABA(B) receptors and spontaneous activity to release probability both in vivo and in vitro in young-adult mice. Maximal activation of presynaptic GABA(B) receptors by baclofen reduced synaptic output by about 45% in whole-cell voltage clamp slice recordings, which was accompanied by a reduction in short-term depression. A similar reduction in transmission was observed when baclofen was applied in vivo by microiontophoresis during juxtacellular recordings using piggyback electrodes. No significant change in synaptic transmission was observed during application of the GABA(B) receptor antagonist CGP54626 both during in vivo and slice recordings, suggesting a low ambient GABA concentration. Interestingly, we observed that synapses with a high spontaneous frequency showed almost no synaptic depression during auditory stimulation, whereas synapses with a low spontaneous frequency did depress during noise bursts. Our data thus suggest that spontaneous firing can tonically reduce release probability in vivo. In addition, our data show that the ambient GABA concentration in the auditory brainstem is too low to activate the GABA(B) receptor at the calyx of Held significantly, but that activation of GABA(B) receptors can reduce sound-evoked synaptic depression.
Asunto(s)
Potenciales de Acción , Depresión Sináptica a Largo Plazo , Receptores de GABA-B/metabolismo , Sinapsis/fisiología , Estimulación Acústica , Factores de Edad , Animales , Baclofeno/farmacología , Agonistas de Receptores GABA-B/farmacología , Antagonistas de Receptores de GABA-B/farmacología , Ratones , Ratones Endogámicos C57BL , Compuestos Organofosforados/farmacología , Sinapsis/metabolismo , Potenciales Sinápticos , Tegmento Mesencefálico/citología , Tegmento Mesencefálico/metabolismo , Tegmento Mesencefálico/fisiologíaRESUMEN
GABAB receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. GABAB receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca2+ influx through NMDA receptors when activated by spillover of GABA from neighboring GABAergic terminals. Here, we show that an excitatory signaling cascade enables spines to counteract this GABAB-mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamin-dependent endocytosis of GABAB receptors. NMDA-dependent internalization of GABAB receptors requires activation of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), which associates with GABAB receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABAB1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABAB receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABAB receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABAB1b subunit isoform, which is the isoform that clusters with inhibitory effector K+ channels in the spines. Consistent with this, NMDA receptor activation in neurons impairs the ability of GABAB receptors to activate K+ channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABAB receptors through CaMKII-mediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABAB receptors.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Receptores de GABA-B/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/química , Células Cultivadas , Ratones , Ratones Noqueados , Fosforilación , Ratas , Receptores de GABA-B/deficiencia , Serina/genética , Serina/metabolismoRESUMEN
GABAB receptors are G-protein coupled receptors for the inhibitory neurotransmitter GABA. Functional GABAB receptors are formed as heteromers of GABAB1 and GABAB2 subunits, which further associate with various regulatory and signaling proteins to provide receptor complexes with distinct pharmacological and physiological properties. GABAB receptors are widely distributed in nervous tissue, where they are involved in a number of processes and in turn are subject to a number of regulatory mechanisms. In this review, we summarize current knowledge of the cellular distribution and function of the receptors in the inner ear and auditory pathway of the mammalian brainstem and midbrain. The findings suggest that in these regions, GABAB receptors are involved in processes essential for proper auditory function, such as cochlear amplifier modulation, regulation of spontaneous activity, binaural and temporal information processing, and predictive coding. Since impaired GABAergic inhibition has been found to be associated with various forms of hearing loss, GABAB dysfunction could also play a role in some pathologies of the auditory system.
Asunto(s)
Sordera , Receptores de GABA-B , Animales , Membrana Celular , Cognición , Mamíferos , Ácido gamma-AminobutíricoRESUMEN
The cranial window technique has proven to be an effective method for in vivo imaging of cortical activity. However, given the invasive nature of this procedure, possible side effects could be expected in the nervous system. In this study, we evaluated the effects of unilateral cranial window surgery on auditory function in C57BL6 mice using electrophysiological and behavioral approaches. We found that one week after implantation, mice exhibited both increased thresholds and decreased amplitudes of their auditory brainstem responses. These changes were accompanied by a decrease in distortion product otoacoustic emissions, indicating a deterioration in cochlear function. In addition, behavioral testing of these mice revealed reduced suppression of their acoustic startle response by gap prepulse, suggesting a deficit in auditory processing or possibly the presence of tinnitus. The changes in auditory function appeared to be only partially reversible within four weeks after surgery. Thus, our findings suggest that cranial window implantation causes long-term functional changes in the auditory system that should be considered when interpreting data from optical imaging techniques.
Asunto(s)
Audición , Reflejo de Sobresalto , Animales , Ratones , Ratones Endogámicos C57BL , Prótesis e Implantes , Potenciales Evocados Auditivos del Tronco EncefálicoRESUMEN
Noise-induced tinnitus is generally associated with hearing impairment caused by traumatic acoustic overexposure. Previous studies in laboratory animals and human subjects, however, have observed differences in tinnitus susceptibility, even among individuals with similar hearing loss. The mechanisms underlying increased sensitivity or, conversely, resistance to tinnitus are still incompletely understood. Here, we used behavioral tests and ABR audiometry to compare the sound-evoked responses of mice that differed in the presence of noise-induced tinnitus. The aim was to find a specific pre-exposure neurophysiological marker that would predict the development of tinnitus after acoustic trauma. Noise-exposed mice were screened for tinnitus-like behavior with the GPIAS paradigm and subsequently divided into tinnitus (+T) and non-tinnitus (-T) groups. Both groups showed hearing loss after exposure, manifested by elevated audiometric thresholds along with reduced amplitudes and prolonged latencies of ABR waves. Prior to exposure, except for a slightly increased slope of growth function for ABR amplitudes in +T mice, the two groups did not show significant audiometric differences. Behavioral measures, such as the magnitude of the acoustic startle response and its inhibition by gap pre-pulse, were also similar before exposure in both groups. However, +T mice showed significantly increased suppression of the acoustic startle response in the presence of background noise of moderate intensity. Thus, increased modulation of startle by background sounds may represent a behavioral correlate of susceptibility to noise-induced tinnitus, and its measurement may form the basis of a simple non-invasive method for predicting tinnitus development in laboratory rodents.
RESUMEN
Stress responses are activated by the hypothalamic-pituitary-adrenal axis (HPA axis), culminating in the release of glucocorticoids. During prolonged periods of secretion of glucocorticoids or inappropriate behavioral responses to a stressor, pathologic conditions may occur. Increased glucocorticoid concentration is linked to generalized anxiety, and there are knowledge gaps regarding its regulation. It is known that the HPA axis is under GABAergic control, but the contribution of the individual subunits of the GABA receptor is largely unknown. In this study, we investigated the relationship between the α5 subunit and corticosterone levels in a new mouse model deficient for Gabra5, which is known to be linked to anxiety disorders in humans and phenologs observed in mice. We observed decreased rearing behavior, suggesting lower anxiety in the Gabra5-/- animals; however, such a phenotype was absent in the open field and elevated plus maze tests. In addition to decreased rearing behavior, we also found decreased levels of fecal corticosterone metabolites in Gabra5-/- mice indicating a lowered stress response. Moreover, based on the electrophysiological recordings where we observed a hyperpolarized state of hippocampal neurons, we hypothesize that the constitutive ablation of the Gabra5 gene leads to functional compensation with other channels or GABA receptor subunits in this model.
Asunto(s)
Corticosterona , Glucocorticoides , Humanos , Ratones , Animales , Sistema Hipotálamo-Hipofisario/metabolismo , Sistema Hipófiso-Suprarrenal/metabolismo , Ansiedad , Receptores de GABA/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.
Asunto(s)
Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Receptores de GABA-B/metabolismo , Sinapsis/metabolismo , Animales , Northern Blotting , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/ultraestructura , Inmunohistoquímica , Memoria/fisiología , Ratones , Ratones Mutantes , Microscopía Confocal , Microscopía Electrónica de Transmisión , Isoformas de Proteínas/genética , Receptores de GABA-B/genética , Sinapsis/ultraestructura , TransfecciónRESUMEN
GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed that GABA(B(1a,2)) receptors are more abundant than GABA(B(1b,2)) receptors at glutamatergic terminals. Accordingly, it was found that GABA(B(1a,2)) receptors are more efficient than GABA(B(1b,2)) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA(B(1a,2)) and GABA(B(1b,2)) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA(B1a) than GABA(B1b) protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations >or=1 microm, both GABA(B(1a,2)) and GABA(B(1b,2)) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA(B(1a,2)) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA(B(1a,2)) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA(B(1a,2)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance.
Asunto(s)
Depresión Sináptica a Largo Plazo/fisiología , Fibras Musgosas del Hipocampo/fisiología , Receptores de GABA-B/fisiología , Transmisión Sináptica/fisiología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fibras Musgosas del Hipocampo/química , Isoformas de Proteínas/fisiologíaRESUMEN
Inhibitory circuits in the auditory brainstem undergo multiple postnatal changes that are both activity-dependent and activity-independent. We tested to see if the shift from GABA- to glycinergic transmission, which occurs in the rat medial nucleus of the trapezoid body (MNTB) around the onset of hearing, depends on sound-evoked neuronal activity. We prevented the activity by bilateral cochlear ablations in early postnatal rats and studied ionotropic GABA and glycine receptors in MNTB neurons after hearing onset. The removal of the cochlea decreased responses of GABAA and glycine receptors to exogenous agonists as well as the amplitudes of inhibitory postsynaptic currents. The reduction was accompanied by a decrease in the number of glycine receptor- or vesicular GABA transporter-immunopositive puncta. Furthermore, the ablations markedly affected the switch in presynaptic GABAA to glycine receptors. The increase in the expression of postsynaptic glycine receptors and the shift in inhibitory transmitters were not prevented. The results suggest that inhibitory transmission in the MNTB is subject to multiple developmental signals and support the idea that auditory experience plays a role in the maturation of the brainstem glycinergic circuits.
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Técnicas de Ablación , Cóclea/fisiopatología , Cóclea/cirugía , Inhibición Neural/fisiología , Transmisión Sináptica , Cuerpo Trapezoide/fisiología , Animales , Animales Recién Nacidos , Potenciales Evocados Auditivos del Tronco Encefálico/fisiología , Agonistas de Receptores de GABA-A/farmacología , Potenciales Postsinápticos Inhibidores/fisiología , Masculino , Inhibición Neural/efectos de los fármacos , Ratas , Receptores de GABA-A/fisiología , Receptores de Glicina/agonistas , Receptores de Glicina/metabolismo , Receptores de Glicina/fisiología , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
GABAB receptors (GBRs) are key regulators of synaptic release but little is known about trafficking mechanisms that control their presynaptic abundance. We now show that sequence-related epitopes in APP, AJAP-1 and PIANP bind with nanomolar affinities to the N-terminal sushi-domain of presynaptic GBRs. Of the three interacting proteins, selectively the genetic loss of APP impaired GBR-mediated presynaptic inhibition and axonal GBR expression. Proteomic and functional analyses revealed that APP associates with JIP and calsyntenin proteins that link the APP/GBR complex in cargo vesicles to the axonal trafficking motor. Complex formation with GBRs stabilizes APP at the cell surface and reduces proteolysis of APP to Aß, a component of senile plaques in Alzheimer's disease patients. Thus, APP/GBR complex formation links presynaptic GBR trafficking to Aß formation. Our findings support that dysfunctional axonal trafficking and reduced GBR expression in Alzheimer's disease increases Aß formation.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Amiloide/metabolismo , Transporte Axonal , Receptores de GABA-B/metabolismo , Secuencia de Aminoácidos , Péptidos beta-Amiloides/química , Animales , Axones/metabolismo , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/metabolismo , Membrana Celular/metabolismo , Dendritas/metabolismo , Epítopos/metabolismo , Proteínas de Unión al GTP/metabolismo , Células HEK293 , Humanos , Cinesinas/metabolismo , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica , Estabilidad Proteica , Proteómica , Transducción de Señal , Sinapsis/metabolismoRESUMEN
Rimonabant is a potent and selective cannabinoid CB1 receptor antagonist widely used in animal and clinical studies. Besides its antagonistic properties, numerous studies have shown that, at micromolar concentrations rimonabant behaves as an inverse agonist at CB1 receptors. The mechanism underpinning this activity is unclear. Here we show that micromolar concentrations of rimonabant inhibited Gαi/o-type G proteins, resulting in a receptor-independent block of G protein signaling. Accordingly, rimonabant decreased basal and agonist stimulated [35S]GTPγS binding to cortical membranes of CB1- and GABAB-receptor KO mice and Chinese Hamster Ovary (CHO) cell membranes stably transfected with GABAB or D2 dopamine receptors. The structural analog of rimonabant, AM251, decreased basal and baclofen-stimulated GTPγS binding to rat cortical and CHO cell membranes expressing GABAB receptors. Rimonabant prevented G protein-mediated GABAB and D2 dopamine receptor signaling to adenylyl cyclase in Human Embryonic Kidney 293â¯cells and to G protein-coupled inwardly rectifying K+ channels (GIRK) in midbrain dopamine neurons of CB1 KO mice. Rimonabant suppressed GIRK gating induced by GTPγS in CHO cells transfected with GIRK, consistent with a receptor-independent action. Bioluminescent resonance energy transfer (BRET) measurements in living CHO cells showed that, in presence or absence of co-expressed GABAB receptors, rimonabant stabilized the heterotrimeric Gαi/o-protein complex and prevented conformational rearrangements induced by GABAB receptor activation. Rimonabant failed to inhibit Gαs-mediated signaling, supporting its specificity for Gαi/o-type G proteins. The inhibition of Gαi/o protein provides a new site of rimonabant action that may help to understand its pharmacological and toxicological effects occurring at high concentrations.
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Antagonistas de Receptores de Cannabinoides/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Cricetulus , Agonistas de Receptores GABA-B/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Modelos Biológicos , Unión Proteica/efectos de los fármacos , Ratas , Receptor Cannabinoide CB1/genética , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Rimonabant , Transducción de Señal/efectos de los fármacosRESUMEN
The mammalian medial nucleus of the trapezoid body (MNTB) harbors one of the most powerful terminals in the CNS, the calyx of Held. The mechanisms known to regulate this synaptic relay are relatively ineffective. Here, we report the presence of a remarkably robust and fast-acting glycinergic inhibitory system capable of suppressing calyceal transmission. Evoked glycinergic IPSCs were relatively small in 2-week-old rats, an age by which calyceal maturation has reportedly neared completion. However, by postnatal day 25 (P25), glycinergic transmission had undergone a vigorous transformation, resulting in peak synaptic conductances as high as 280 nS. These are comparable with glutamatergic conductances activated by calyceal inputs. Decay kinetics for IPSCs were severalfold faster than for glycinergic synaptic events reported previously. At physiological temperatures in P25 rats, IPSCs decayed in approximately 1 msec and could be elicited at frequencies up to 500 Hz. Moreover, EPSPs triggered by glutamatergic signals derived from the calyx or simulated by conductance clamp were suppressed when preceded by simulated glycinergic IPSPs. The matching of excitatory transmission in the calyx of Held by a powerful, precision inhibitory system suggests that the relay function of the MNTB may be rapidly modified during sound localization.
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Vías Auditivas/fisiología , Tronco Encefálico/fisiología , Inhibición Neural/fisiología , Neuronas/fisiología , Sinapsis/fisiología , Animales , Tronco Encefálico/citología , Tronco Encefálico/efectos de los fármacos , Estimulación Eléctrica/métodos , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Antagonistas del GABA/farmacología , Ácido Glutámico/metabolismo , Glicina/metabolismo , Glicinérgicos/farmacología , Técnicas In Vitro , Inhibición Neural/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Ratas , Localización de Sonidos/fisiología , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , TemperaturaRESUMEN
GABA(B) receptors (GABA(B)Rs) regulate the excitability of most neurons in the central nervous system by modulating the activity of enzymes and ion channels. In the sustained presence of the neurotransmitter γ-aminobutyric acid, GABA(B)Rs exhibit a time-dependent decrease in the receptor response-a phenomenon referred to as homologous desensitization. Desensitization prevents excessive receptor influences on neuronal activity. Much work focused on the mechanisms of GABA(B)R desensitization that operate at the receptor and control receptor expression at the plasma membrane. Over the past few years, it became apparent that GABA(B)Rs additionally evolved mechanisms for faster desensitization. These mechanisms operate at the G protein rather than at the receptor and inhibit G protein signaling within seconds of agonist exposure. The mechanisms for fast desensitization are ideally suited to regulate receptor-activated ion channel responses, which influence neuronal activity on a faster timescale than effector enzymes. Here, we provide an update on the mechanisms for fast desensitization of GABA(B)R responses and discuss physiological and pathophysiological implications.
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Neuronas/metabolismo , Receptores de GABA-B/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Membrana Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Transducción de Señal , Factores de TiempoRESUMEN
The physiological functions of glycine receptors (GlyRs) depend on their subcellular locations. In axonal terminals of the central neurons, GlyRs trigger a slow facilitation of presynaptic transmitter release; however, their spatial relationship to the release sites is not known. In this study, we examined the distribution of GlyRs in the rat glutamatergic calyx of Held nerve terminal using high-resolution pre-embedding immunoelectron microscopy. We performed a quantitative analysis of GlyR-associated immunogold (IG) labeling in 3D reconstructed calyceal segments. A variable density of IG particles and their putative accumulations, inferred from the frequency distribution of inter-IG distances, indicated a non-uniform distribution of the receptors in the calyx. Subsequently, increased densities of IG particles were found in calyceal swellings, structures characterized by extensive exocytosis of glutamate. In swellings as well as in larger calyceal stalks, IG particles did not tend to accumulate near the glutamate releasing zones. On the other hand, GlyRs in swellings (but not in stalks) preferentially occupied membrane regions, unconnected to postsynaptic cells and presumably accessible by ambient glycine. Furthermore, the sites with increased GlyR concentrations were found in swellings tightly juxtaposed with GABA/glycinergic nerve endings. Thus, the results support the concept of an indirect mechanism underlying the modulatory effects of calyceal GlyRs, activated by glycine spillover. We also suggest the existence of an activity-dependent mechanism regulating the surface distribution of α homomeric GlyRs in axonal terminals of central neurons.