RESUMO
Sensitization of the capsaicin receptor TRPV1 is central to the initiation of pathological forms of pain, and multiple signaling cascades are known to enhance TRPV1 activity under inflammatory conditions. How might detrimental escalation of TRPV1 activity be counteracted? Using a genetic-proteomic approach, we identify the GABAB1 receptor subunit as bona fide inhibitor of TRPV1 sensitization in the context of diverse inflammatory settings. We find that the endogenous GABAB agonist, GABA, is released from nociceptive nerve terminals, suggesting an autocrine feedback mechanism limiting TRPV1 sensitization. The effect of GABAB on TRPV1 is independent of canonical G protein signaling and rather relies on close juxtaposition of the GABAB1 receptor subunit and TRPV1. Activating the GABAB1 receptor subunit does not attenuate normal functioning of the capsaicin receptor but exclusively reverts its sensitized state. Thus, harnessing this mechanism for anti-pain therapy may prevent adverse effects associated with currently available TRPV1 blockers.
Assuntos
Comunicação Autócrina , Neurônios/metabolismo , Dor/metabolismo , Receptores de GABA-B/metabolismo , Canais de Cátion TRPV/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Células Cultivadas , Retroalimentação , Feminino , Masculino , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
GABAB receptors (GBRs), the G protein-coupled receptors for GABA, regulate synaptic transmission throughout the brain. A main synaptic function of GBRs is the gating of Cav2.2-type Ca2+ channels. However, the cellular compartment where stable GBR/Cav2.2 signaling complexes form remains unknown. In this study, we demonstrate that the vesicular protein synaptotagmin-11 (Syt11) binds to both the auxiliary GBR subunit KCTD16 and Cav2.2 channels. Through these dual interactions, Syt11 recruits GBRs and Cav2.2 channels to post-Golgi vesicles, thus facilitating assembly of GBR/Cav2.2 signaling complexes. In addition, Syt11 stabilizes GBRs and Cav2.2 channels at the neuronal plasma membrane by inhibiting constitutive internalization. Neurons of Syt11 knockout mice exhibit deficits in presynaptic GBRs and Cav2.2 channels, reduced neurotransmitter release, and decreased GBR-mediated presynaptic inhibition, highlighting the critical role of Syt11 in the assembly and stable expression of GBR/Cav2.2 complexes. These findings support that Syt11 acts as a vesicular scaffold protein, aiding in the assembly of signaling complexes from low-abundance components within transport vesicles. This mechanism enables insertion of pre-assembled functional signaling units into the synaptic membrane.
Assuntos
Camundongos Knockout , Transdução de Sinais , Sinaptotagminas , Animais , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Camundongos , Humanos , Neurônios/metabolismo , Transmissão Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminações Pré-Sinápticas/metabolismo , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/genética , Complexo de Golgi/metabolismo , Ligação Proteica , Células HEK293RESUMO
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.
Assuntos
Canais de Potássio/genética , Canais de Potássio/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Animais , Química Encefálica/genética , Células CHO , Cricetinae , Cricetulus , Fenômenos Eletrofisiológicos/genética , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Cinética , Masculino , Camundongos , Camundongos Knockout , Técnicas de Patch-Clamp , Receptores Acoplados a Proteínas G/metabolismo , Receptores KIR/metabolismoRESUMO
Cholecystokinin-expressing interneurons (CCK-INs) mediate behavior state-dependent inhibition in cortical circuits and themselves receive strong GABAergic input. However, it remains unclear to what extent GABAB receptors (GABABRs) contribute to their inhibitory control. Using immunoelectron microscopy, we found that CCK-INs in the rat hippocampus possessed high levels of dendritic GABABRs and KCTD12 auxiliary proteins, whereas postsynaptic effector Kir3 channels were present at lower levels. Consistently, whole-cell recordings revealed slow GABABR-mediated inhibitory postsynaptic currents (IPSCs) in most CCK-INs. In spite of the higher surface density of GABABRs in CCK-INs than in CA1 principal cells, the amplitudes of IPSCs were comparable, suggesting that the expression of Kir3 channels is the limiting factor for the GABABR currents in these INs. Morphological analysis showed that CCK-INs were diverse, comprising perisomatic-targeting basket cells (BCs), as well as dendrite-targeting (DT) interneurons, including a previously undescribed DT type. GABABR-mediated IPSCs in CCK-INs were large in BCs, but small in DT subtypes. In response to prolonged activation, GABABR-mediated currents displayed strong desensitization, which was absent in KCTD12-deficient mice. This study highlights that GABABRs differentially control CCK-IN subtypes, and the kinetics and desensitization of GABABR-mediated currents are modulated by KCTD12 proteins.
Assuntos
Colecistocinina/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/metabolismo , Canais de Potássio/metabolismo , Receptores de GABA-A/metabolismo , Animais , Região CA1 Hipocampal/metabolismo , Região CA1 Hipocampal/ultraestrutura , Dendritos/metabolismo , Dendritos/ultraestrutura , Imuno-Histoquímica , Interneurônios/ultraestrutura , Masculino , Microscopia Imunoeletrônica , Técnicas de Patch-Clamp , Ratos Wistar , Técnicas de Cultura de TecidosRESUMO
Stabilization of neuronal activity by homeostatic control systems is fundamental for proper functioning of neural circuits. Failure in neuronal homeostasis has been hypothesized to underlie common pathophysiological mechanisms in a variety of brain disorders. However, the key molecules regulating homeostasis in central mammalian neural circuits remain obscure. Here, we show that selective inactivation of GABAB, but not GABA(A), receptors impairs firing rate homeostasis by disrupting synaptic homeostatic plasticity in hippocampal networks. Pharmacological GABA(B) receptor (GABA(B)R) blockade or genetic deletion of the GB(1a) receptor subunit disrupts homeostatic regulation of synaptic vesicle release. GABA(B)Rs mediate adaptive presynaptic enhancement to neuronal inactivity by two principle mechanisms: First, neuronal silencing promotes syntaxin-1 switch from a closed to an open conformation to accelerate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and second, it boosts spike-evoked presynaptic calcium flux. In both cases, neuronal inactivity removes tonic block imposed by the presynaptic, GB(1a)-containing receptors on syntaxin-1 opening and calcium entry to enhance probability of vesicle fusion. We identified the GB(1a) intracellular domain essential for the presynaptic homeostatic response by tuning intermolecular interactions among the receptor, syntaxin-1, and the Ca(V)2.2 channel. The presynaptic adaptations were accompanied by scaling of excitatory quantal amplitude via the postsynaptic, GB(1b)-containing receptors. Thus, GABA(B)Rs sense chronic perturbations in GABA levels and transduce it to homeostatic changes in synaptic strength. Our results reveal a novel role for GABA(B)R as a key regulator of population firing stability and propose that disruption of homeostatic synaptic plasticity may underlie seizure's persistence in the absence of functional GABA(B)Rs.
Assuntos
Hipocampo/fisiologia , Homeostase , Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Animais , Células Cultivadas , Potenciais Evocados , Hipocampo/citologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Adult neurogenesis is tightly regulated through the interaction of neural stem/progenitor cells (NSCs) with their niche. Neurotransmitters, including GABA activation of GABAA receptor ion channels, are important niche signals. We show that adult mouse hippocampal NSCs and their progeny express metabotropic GABAB receptors. Pharmacological inhibition of GABAB receptors stimulated NSC proliferation and genetic deletion of GABAB1 receptor subunits increased NSC proliferation and differentiation of neuroblasts in vivo. Cell-specific conditional deletion of GABAB receptors supports a cell-autonomous role in newly generated cells. Our data indicate that signaling through GABAB receptors is an inhibitor of adult neurogenesis.
Assuntos
Hipocampo/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Receptores de GABA-B/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/genética , Antagonistas de Receptores de GABA-B/farmacologia , Hipocampo/citologia , Camundongos , Camundongos Knockout , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Neurônios/citologia , Neurônios/metabolismo , Compostos Organofosforados/farmacologia , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Receptores de GABA-B/genética , Transdução de Sinais/fisiologiaRESUMO
GABA(B) receptors (GABA(B)Rs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the CNS. In the past 5 years, notable advances have been made in our understanding of the molecular composition of these receptors. GABA(B)Rs are now known to comprise principal and auxiliary subunits that influence receptor properties in distinct ways. The principal subunits regulate the surface expression and the axonal versus dendritic distribution of these receptors, whereas the auxiliary subunits determine agonist potency and the kinetics of the receptor response. This Review summarizes current knowledge on how the subunit composition of GABA(B)Rs affects the distribution of these receptors, neuronal processes and higher brain functions.
Assuntos
Neurônios/metabolismo , Receptores de GABA-B/fisiologia , Animais , Axônios/metabolismo , Axônios/fisiologia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Dendritos/metabolismo , Dendritos/fisiologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Ácido Glutâmico/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Fosforilação , Filogenia , Terminações Pré-Sinápticas/fisiologia , Receptor Cross-Talk/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Ácido gama-Aminobutírico/fisiologiaRESUMO
Stressful life events increase the susceptibility to developing psychiatric disorders such as depression; however, many individuals are resilient to such negative effects of stress. Determining the neurobiology underlying this resilience is instrumental to the development of novel and more effective treatments for stress-related psychiatric disorders. GABAB receptors are emerging therapeutic targets for the treatment of stress-related disorders such as depression. These receptors are predominantly expressed as heterodimers of a GABAB(2) subunit with either a GABAB(1a) or a GABAB(1b) subunit. Here we show that mice lacking the GABAB(1b) receptor isoform are more resilient to both early-life stress and chronic psychosocial stress in adulthood, whereas mice lacking GABAB(1a) receptors are more susceptible to stress-induced anhedonia and social avoidance compared with wild-type mice. In addition, increased hippocampal expression of the GABAB(1b) receptor subunit is associated with a depression-like phenotype in the helpless H/Rouen genetic mouse model of depression. Stress resilience in GABAB(1b)(-/-) mice is coupled with increased proliferation and survival of newly born cells in the adult ventral hippocampus and increased stress-induced c-Fos activation in the hippocampus following early-life stress. Taken together, the data suggest that GABAB(1) receptor subunit isoforms differentially regulate the deleterious effects of stress and, thus, may be important therapeutic targets for the treatment of depression.
Assuntos
Depressão/metabolismo , Receptores de GABA-B/fisiologia , Anedonia , Animais , Comportamento Animal , Proliferação de Células , Corticosterona/metabolismo , Depressão/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Isoformas de Proteínas/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Risco , Estresse Psicológico , NataçãoRESUMO
Underwater radiated noise from merchant ships was measured opportunistically from multiple spatial aspects to estimate signature source levels and directionality. Transiting ships were tracked via the Automatic Identification System in a shipping lane while acoustic pressure was measured at the ships' keel and beam aspects. Port and starboard beam aspects were 15°, 30°, and 45° in compliance with ship noise measurements standards [ANSI/ASA S12.64 (2009) and ISO 17208-1 (2016)]. Additional recordings were made at a 10° starboard aspect. Source levels were derived with a spherical propagation (surface-affected) or a modified Lloyd's mirror model to account for interference from surface reflections (surface-corrected). Ship source depths were estimated from spectral differences between measurements at different beam aspects. Results were exemplified with a 4870 and a 10 036 twenty-foot equivalent unit container ship at 40%-56% and 87% of service speeds, respectively. For the larger ship, opportunistic ANSI/ISO broadband levels were 195 (surface-affected) and 209 (surface-corrected) dB re 1 µPa2 1 m. Directionality at a propeller blade rate of 8 Hz exhibited asymmetries in stern-bow (<6 dB) and port-starboard (<9 dB) direction. Previously reported broadband levels at 10° aspect from McKenna, Ross, Wiggins, and Hildebrand [(2012b). J. Acoust. Soc. Am. 131, 92-103] may be â¼12 dB lower than respective surface-affected ANSI/ISO standard derived levels.
RESUMO
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.
Assuntos
Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Condutividade Elétrica , Agonistas dos Receptores de GABA-B , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Cinética , Camundongos , Neurônios/metabolismo , Oócitos/metabolismo , Potássio/metabolismo , Canais de Potássio/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Transdução de Sinais , XenopusRESUMO
G protein-coupled receptors (GPCRs) have key roles in cell-cell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA(B) receptor is one of the most abundant, being distributed in most brain regions, on either pre- or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA(B) receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA(B1) subunits. Destabilizing the oligomers using a competitor or a GABA(B1) mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA(B) receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA(B) heterodimers.
Assuntos
Receptores de GABA-B/química , Transdução de Sinais/fisiologia , Regulação Alostérica/genética , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Mutagênese Sítio-Dirigida , Isoformas de Proteínas/química , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Multimerização Proteica/genética , Estabilidade Proteica , Receptores de GABA-B/deficiência , Receptores de GABA-B/genética , Transdução de Sinais/genéticaRESUMO
Cuvier's beaked whales (Ziphius cavirostris) were tracked using two volumetric small-aperture (â¼1 m element spacing) hydrophone arrays, embedded into a large-aperture (â¼1 km element spacing) seafloor hydrophone array of five nodes. This array design can reduce the minimum number of nodes that are needed to record the arrival of a strongly directional echolocation sound from 5 to 2, while providing enough time-differences of arrivals for a three-dimensional localization without depending on any additional information such as multipath arrivals. To illustrate the capabilities of this technique, six encounters of up to three Cuvier's beaked whales were tracked over a two-month recording period within an area of 20 km(2) in the Southern California Bight. Encounter periods ranged from 11 min to 33 min. Cuvier's beaked whales were found to reduce the time interval between echolocation clicks while alternating between two inter-click-interval regimes during their descent towards the seafloor. Maximum peak-to-peak source levels of 179 and 224 dB re 1 µPa @ 1 m were estimated for buzz sounds and on-axis echolocation clicks (directivity index = 30 dB), respectively. Source energy spectra of the on-axis clicks show significant frequency components between 70 and 90 kHz, in addition to their typically noted FM upsweep at 40-60 kHz.
Assuntos
Acústica/instrumentação , Ecolocação , Biologia Marinha/instrumentação , Baleias/fisiologia , Algoritmos , Animais , Comportamento Animal , Mergulho , Oceano Pacífico , Espectrografia do Som , Transdutores de PressãoRESUMO
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.
Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Potássio/metabolismo , Multimerização Proteica/fisiologia , Receptores de GABA-B/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Membrana Celular/genética , Membrana Celular/metabolismo , Chlorocebus aethiops , Hipocampo/citologia , Camundongos , Camundongos Knockout , Neurônios/citologia , Canais de Potássio/genética , Receptores de GABA-B/genéticaRESUMO
Serotonin (5-HT)2C receptors play a role in psychoaffective disorders and often contribute to the antidepressant and anxiolytic effects of psychotropic drugs. During stress, activation of these receptors exerts a negative feedback on 5-HT release, probably by increasing the activity of GABAergic interneurons. However, to date, the GABA receptor types that mediate the 5-HT2C receptor-induced feedback inhibition are still unknown. To address this question, we assessed the inhibition of 5-HT turnover by a 5-HT2C receptor agonist (RO 60-0175) at the hippocampal level and under conditions of stress, after pharmacological or genetic inactivation of either GABA-A or GABA-B receptors in mice. Neither the GABA-B receptor antagonist phaclofen nor the specific genetic ablation of either GABA-B1a or GABA-B1b subunits altered the inhibitory effect of RO 60-0175, although 5-HT turnover was markedly decreased in GABA-B1a knock-out mice in both basal and stress conditions. In contrast, the 5-HT2C receptor-mediated inhibition of 5-HT turnover was reduced by the GABA-A receptor antagonist bicuculline. However, a significant effect of 5-HT2C receptor activation persisted in mutant mice deficient in the α3 subunit of GABA-A receptors. It can be inferred that non-α3 subunit-containing GABA-A receptors, but not GABA-B receptors, mediate the 5-HT2C -induced inhibition of stress-induced increase in hippocampal 5-HT turnover in mice.
Assuntos
GABAérgicos/farmacologia , Receptor 5-HT2C de Serotonina/metabolismo , Receptores de GABA/genética , Estresse Psicológico/genética , Estresse Psicológico/metabolismo , Animais , Modelos Animais de Doenças , Etilaminas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Indóis/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de GABA/deficiência , Serotonina/metabolismo , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/patologiaRESUMO
Opportunistic observations of behavioral responses by delphinids to incidental mid-frequency active (MFA) sonar were recorded in the Southern California Bight from 2004 through 2008 using visual focal follows, static hydrophones, and autonomous recorders. Sound pressure levels were calculated between 2 and 8 kHz. Surface behavioral responses were observed in 26 groups from at least three species of 46 groups out of five species encountered during MFA sonar incidents. Responses included changes in behavioral state or direction of travel, changes in vocalization rates and call intensity, or a lack of vocalizations while MFA sonar occurred. However, 46% of focal groups not exposed to sonar also changed their behavior, and 43% of focal groups exposed to sonar did not change their behavior. Mean peak sound pressure levels when a behavioral response occurred were around 122 dB re: 1 µPa. Acoustic localizations of dolphin groups exhibiting a response gave insight into nighttime movement patterns and provided evidence that impacts of sonar may be mediated by behavioral state. The lack of response in some cases may indicate a tolerance of or habituation to MFA sonar by local populations; however, the responses that occur at lower received levels may point to some sensitization as well.
Assuntos
Percepção Auditiva , Comportamento Animal , Golfinhos/psicologia , Meio Ambiente , Ruído , Ultrassom/métodos , Acústica , Animais , Golfinhos/classificação , Golfinhos/fisiologia , Exposição Ambiental , Comportamento Alimentar , Pressão , Comportamento Social , Espectrografia do Som , Natação , Fatores de Tempo , Vocalização AnimalRESUMO
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.
RESUMO
Adherens junction-associated protein 1 (AJAP1) has been implicated in brain diseases; however, a pathogenic mechanism has not been identified. AJAP1 is widely expressed in neurons and binds to γ-aminobutyric acid type B receptors (GBRs), which inhibit neurotransmitter release at most synapses in the brain. Here, we show that AJAP1 is selectively expressed in dendrites and trans-synaptically recruits GBRs to presynaptic sites of neurons expressing AJAP1. We have identified several monoallelic AJAP1 variants in individuals with epilepsy and/or neurodevelopmental disorders. Specifically, we show that the variant p.(W183C) lacks binding to GBRs, resulting in the inability to recruit them. Ultrastructural analysis revealed significantly decreased presynaptic GBR levels in Ajap1-/- and Ajap1W183C/+ mice. Consequently, these mice exhibited reduced GBR-mediated presynaptic inhibition at excitatory and inhibitory synapses, along with impaired synaptic plasticity. Our study reveals that AJAP1 enables the postsynaptic neuron to regulate the level of presynaptic GBR-mediated inhibition, supporting the clinical relevance of loss-of-function AJAP1 variants.
Assuntos
Neurotransmissores , Sinapses , Transmissão Sináptica , Animais , Feminino , Humanos , Masculino , Camundongos , Alelos , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/patologia , Mutação com Perda de Função , Camundongos Knockout , Transtornos do Neurodesenvolvimento/metabolismo , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Plasticidade Neuronal , Neurônios/metabolismo , Neurotransmissores/metabolismo , Sinapses/metabolismo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismoRESUMO
GABA(B) receptors assemble from principle and auxiliary subunits. The principle subunits GABA(B1) and GABA(B2) form functional heteromeric GABA(B(1,2)) receptors that associate with homotetramers of auxiliary KCTD8, -12, -12b, or -16 (named after their K(+) channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and -12b generate desensitizing receptor responses while KCTD8 and -16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABA(B2), and a H1 homology domain. KCTD8 and -16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and -12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and -16. In addition, the H2 domains in KCTD8 and -16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and -12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.
Assuntos
Evolução Molecular , Subunidades Proteicas/metabolismo , Proteínas/metabolismo , Receptores de GABA-B/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Células HEK293 , Humanos , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Proteínas/genética , Receptores de GABA-B/genéticaRESUMO
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.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Receptores de GABA-B/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sequência de Aminoácidos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/química , Células Cultivadas , Camundongos , Camundongos Knockout , Fosforilação , Ratos , Receptores de GABA-B/deficiência , Serina/genética , Serina/metabolismoRESUMO
To study delphinid near surface movements and behavior, two L-shaped hydrophone arrays and one vertical hydrophone line array were deployed at shallow depths (<125 m) from the floating instrument platform R/P FLIP, moored northwest of San Clemente Island in the Southern California Bight. A three-dimensional propagation-model based passive acoustic tracking method was developed and used to track a group of five offshore killer whales (Orcinus orca) using their emitted clicks. In addition, killer whale pulsed calls and high-frequency modulated (HFM) signals were localized using other standard techniques. Based on these tracks sound source levels for the killer whales were estimated. The peak to peak source levels for echolocation clicks vary between 170-205 dB re 1 µPa @ 1 m, for HFM calls between 185-193 dB re 1 µPa @ 1 m, and for pulsed calls between 146-158 dB re 1 µPa @ 1 m.