RESUMEN
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.
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Neurotransmisores , Sinapsis , Transmisión Sináptica , Animales , Femenino , Humanos , Masculino , Ratones , Alelos , Epilepsia/metabolismo , Epilepsia/genética , Epilepsia/patología , Mutación con Pérdida de Función , Ratones Noqueados , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Plasticidad Neuronal , Neuronas/metabolismo , Neurotransmisores/metabolismo , Sinapsis/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismoRESUMEN
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.
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Ratones Noqueados , Transducción de Señal , Sinaptotagminas , Animales , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Ratones , Humanos , Neuronas/metabolismo , Transmisión Sináptica , Receptores de GABA-B/metabolismo , Receptores de GABA-B/genética , Terminales Presinápticos/metabolismo , Canales de Calcio Tipo N/metabolismo , Canales de Calcio Tipo N/genética , Aparato de Golgi/metabolismo , Unión Proteica , Células HEK293RESUMEN
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 metabotropic glutamate receptor 1 (mGlu1) plays a pivotal role in synaptic transmission and neuronal plasticity. Despite the fact that several interacting proteins involved in the mGlu1 subcellular trafficking and intracellular transduction mechanisms have been identified, the protein network associated with this receptor in specific brain areas remains largely unknown. To identify novel mGlu1-associated protein complexes in the mouse cerebellum, we used an unbiased tissue-specific proteomic approach, namely co-immunoprecipitation followed by liquid chromatography/tandem mass spectrometry analysis. Many well-known protein complexes as well as novel interactors were identified, including G-proteins, Homer, δ2 glutamate receptor, 14-3-3 proteins, and Na/K-ATPases. A novel putative interactor, KCTD12, was further investigated. Reverse co-immunoprecipitation with anti-KCTD12 antibodies revealed mGlu1 in wild-type but not in KCTD12-knock-out homogenates. Freeze-fracture replica immunogold labeling co-localization experiments showed that KCTD12 and mGlu1 are present in the same nanodomain in Purkinje cell spines, although at a distance that suggests that this interaction is mediated through interposed proteins. Consistently, mGlu1 could not be co-immunoprecipitated with KCTD12 from a recombinant mammalian cell line co-expressing the two proteins. The possibility that this interaction was mediated via GABAB receptors was excluded by showing that mGlu1 and KCTD12 still co-immunoprecipitated from GABAB receptor knock-out tissue. In conclusion, this study identifies tissue-specific mGlu1-associated protein clusters including KCTD12 at Purkinje cell synapses.
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Proteómica , Receptores de Glutamato Metabotrópico , Ratones , Animales , Células de Purkinje , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de GABA-B/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Glutamatos/metabolismo , Mamíferos/metabolismoRESUMEN
The container shipping line Maersk undertook a Radical Retrofit to improve the energy efficiency of twelve sister container ships. Noise reduction, identified as a potential added benefit of the retrofitting effort, was investigated in this study. A passive acoustic recording dataset from the Santa Barbara Channel off Southern California was used to compile over 100 opportunistic vessel transits of the twelve G-Class container ships, pre- and post-retrofit. Post-retrofit, the G-Class vessels' capacity was increased from ~9,000 twenty-foot equivalent units (TEUs) to ~11,000 TEUs, which required a draft increase of the vessel by 1.5 m on average. The increased vessel draft resulted in higher radiated noise levels (<2 dB) in the mid- and high-frequency bands. Accounting for the Lloyd's mirror (dipole source) effect, the monopole source levels of the post-retrofit ships were found to be significantly lower (>5 dB) than the pre-retrofit ships in the low-frequency band and the reduction was greatest at low speed. Although multiple design changes occurred during retrofitting, the reduction in the low-frequency band most likely results from a reduction in cavitation due to changes in propeller and bow design.
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Ruido , Navíos , Espectrografía del Sonido , AcústicaRESUMEN
Amyloid-ß precursor protein (APP) regulates neuronal activity through the release of secreted APP (sAPP) acting at cell surface receptors. APP and sAPP were reported to bind to the extracellular sushi domain 1 (SD1) of GABAB receptors (GBRs). A 17 amino acid peptide (APP17) derived from APP was sufficient for SD1 binding and shown to mimic the inhibitory effect of sAPP on neurotransmitter release and neuronal activity. The functional effects of APP17 and sAPP were similar to those of the GBR agonist baclofen and blocked by a GBR antagonist. These experiments led to the proposal that sAPP activates GBRs to exert its neuronal effects. However, whether APP17 and sAPP influence classical GBR signaling pathways in heterologous cells was not analyzed. Here, we confirm that APP17 binds to GBRs with nanomolar affinity. However, biochemical and electrophysiological experiments indicate that APP17 does not influence GBR activity in heterologous cells. Moreover, APP17 did not regulate synaptic GBR localization, GBR-activated K+ currents, neurotransmitter release, or neuronal activity in vitro or in vivo. Our results show that APP17 is not a functional GBR ligand and indicate that sAPP exerts its neuronal effects through receptors other than GBRs.
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Péptidos beta-Amiloides , Precursor de Proteína beta-Amiloide , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The synaptic connection from medial habenula (MHb) to interpeduncular nucleus (IPN) is critical for emotion-related behaviors and uniquely expresses R-type Ca2+ channels (Cav2.3) and auxiliary GABAB receptor (GBR) subunits, the K+-channel tetramerization domain-containing proteins (KCTDs). Activation of GBRs facilitates or inhibits transmitter release from MHb terminals depending on the IPN subnucleus, but the role of KCTDs is unknown. We therefore examined the localization and function of Cav2.3, GBRs, and KCTDs in this pathway in mice. We show in heterologous cells that KCTD8 and KCTD12b directly bind to Cav2.3 and that KCTD8 potentiates Cav2.3 currents in the absence of GBRs. In the rostral IPN, KCTD8, KCTD12b, and Cav2.3 co-localize at the presynaptic active zone. Genetic deletion indicated a bidirectional modulation of Cav2.3-mediated release by these KCTDs with a compensatory increase of KCTD8 in the active zone in KCTD12b-deficient mice. The interaction of Cav2.3 with KCTDs therefore scales synaptic strength independent of GBR activation.
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Canales de Calcio Tipo R/metabolismo , Proteínas de Transporte de Catión/metabolismo , Habénula/metabolismo , Terminales Presinápticos/metabolismo , Receptores de GABA/metabolismo , Animales , Canales de Calcio Tipo R/genética , Proteínas de Transporte de Catión/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptores de GABA/genética , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
For a long time metabotropic glutamate receptors (mGluRs) were thought to regulate neuronal functions as obligatory homodimers. Recent reports, however, indicate the existence of heterodimers between group-II and -III mGluRs in the brain, which differ from the homodimers in their signal transduction and sensitivity to negative allosteric modulators (NAMs). Whether the group-I mGluRs, mGlu1 and mGlu5, form functional heterodimers in the brain is still a matter of debate. We now show that mGlu1 and mGlu5 co-purify from brain membranes and hippocampal tissue and co-localize in cultured hippocampal neurons. Complementation assays with mutants deficient in agonist-binding or G protein-coupling reveal that mGlu1/5 heterodimers are functional in heterologous cells and transfected cultured hippocampal neurons. In contrast to heterodimers between group-II and -III mGluRs, mGlu1/5 receptors exhibit a symmetric signal transduction, with both protomers activating G proteins to a similar extent. NAMs of either protomer in mGlu1/5 receptors partially inhibit signaling, showing that both protomers need to be able to reach an active conformation for full receptor activity. Complete heterodimer inhibition is observed when both protomers are locked in their inactive state by a NAM. In summary, our data show that mGlu1/5 heterodimers exhibit a symmetric signal transduction and thus intermediate signaling efficacy and kinetic properties. Our data support the existence of mGlu1/5 heterodimers in neurons and highlight differences in the signaling transduction of heterodimeric mGluRs that influence allosteric modulation.
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Hipocampo/metabolismo , Neuronas/metabolismo , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Regulación Alostérica , Animales , Encéfalo/metabolismo , Cromatografía Liquida , Hipocampo/citología , Ratones , Ratones Noqueados , Multimerización de Proteína , Receptor del Glutamato Metabotropico 5/genética , Receptores de Glutamato Metabotrópico/genética , Transducción de Señal , Espectrometría de Masas en TándemRESUMEN
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
Notch signalling regulates neural stem cell (NSC) proliferation, differentiation and survival for the correct development and functioning of the central nervous system. Overactive Notch2 signalling has been associated with poor prognosis of aggressive brain tumours, such as glioblastoma multiforme (GBM). We recently reported that constitutive expression of the Notch2 intracellular domain (N2ICD) enhances proliferation and gliogenesis in NSCs. Here, we investigated the mechanism by which Notch2 promotes resistance to apoptosis of NSCs to cytotoxic insults. We performed ex vivo studies using NSC cultures from transgenic mice constitutively expressing N2ICD. These NSCs expressed increased levels of pro-survival factors and lack an apoptotic response to the topoisomerase inhibitor etoposide, not showing neither mitochondrial damage nor caspase activation. Interestingly, Notch2 signalling also regulated chemoresistance of human GBM cells to etoposide. We also identified a signalling crosstalk with FGF signalling pathway involved in this resistance to apoptosis of NSCs. Aberrant Notch2 expression enhances fibroblast growth factor receptor-1 (FGFR1) activity to specifically target the AKT-GSK3 signalling pathway to block apoptosis. These results have implications for understanding molecular changes involved in both tumorigenesis and therapy resistance.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Etopósido/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioblastoma/metabolismo , Células-Madre Neurales/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/biosíntesis , Receptor Notch2/metabolismo , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones Transgénicos , Células-Madre Neurales/patología , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor Notch2/genética , Transducción de Señal/genéticaRESUMEN
Following the discovery of GABAB receptors by Norman Bowery and colleagues, cloning and biochemical efforts revealed that GABAB receptors assemble multi-subunit complexes composed of principal and auxiliary subunits. The principal receptor subunits GABAB1a, GABAB1b and GABAB2 form two heterodimeric GABAB(1a,2) and GABAB(1b,2) receptors that can associate with tetramers of auxiliary KCTD (K+ channel tetramerization domain) subunits. Experiments with subunit knock-out mice revealed that GABAB(1b,2) receptors activate slow inhibitory postsynaptic currents (sIPSCs) while GABAB(1a,2) receptors function as heteroreceptors and inhibit glutamate release. Both GABAB(1a,2) and GABAB(1b,2) receptors can serve as autoreceptors and inhibit GABA release. Auxiliary KCTD subunits regulate the duration of sIPSCs and scaffold effector channels at the receptor. GABAB receptors are well known to contribute to thalamic spindle oscillations. Spindles are generated through alternating burst-firing in reciprocally connected glutamatergic thalamocortical relay (TCR) and GABAergic thalamic reticular nucleus (TRN) neurons. The available data implicate postsynaptic GABAB receptors in TCR cells in the regulation of spindle frequency. We now used electrical or optogenetic activation of thalamic spindles and pharmacological experiments in acute slices of knock-out mice to study the impact of GABAB(1a,2) and GABAB(1b,2) receptors on spindle oscillations. We found that selectively GABAB(1a,2) heteroreceptors at TCR to TRN cell synapses regulate oscillation strength, while GABAB(1b,2) receptors control oscillation frequency. The auxiliary subunit KCTD16 influences both oscillation strength and frequency, supporting that KCTD16 regulates network activity through GABAB(1a,2) and GABAB(1b,2) receptors. This article is part of the "Special Issue Dedicated to Norman G. Bowery".
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de GABA-B/metabolismo , Tálamo/metabolismo , Animales , Ácido Glutámico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptores de GABA/deficiencia , Receptores de GABA/genética , Receptores de GABA-B/genética , Sinapsis/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Colecistoquinina/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Potenciales Postsinápticos Inhibidores/fisiología , Interneuronas/metabolismo , Canales de Potasio/metabolismo , Receptores de GABA-A/metabolismo , Animales , Región CA1 Hipocampal/metabolismo , Región CA1 Hipocampal/ultraestructura , Dendritas/metabolismo , Dendritas/ultraestructura , Inmunohistoquímica , Interneuronas/ultraestructura , Masculino , Microscopía Inmunoelectrónica , Técnicas de Placa-Clamp , Ratas Wistar , Técnicas de Cultivo de TejidosRESUMEN
Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and is implicated in the pathophysiology of a number of neuropsychiatric disorders. The GABAB receptors are G-protein coupled receptors consisting of principle subunits and auxiliary potassium channel tetramerization domain (KCTD) subunits. The KCTD subunits 8, 12, 12b and 16 are cytosolic proteins that determine the kinetics of the GABAB receptor response. Previously, we demonstrated that Kctd12 null mutant mice (Kctd12-/-) exhibit increased auditory fear learning and that Kctd12+/- mice show altered circadian activity, as well as increased intrinsic excitability in hippocampal pyramidal neurons. KCTD16 has been demonstrated to influence neuronal excitability by regulating GABAB receptor-mediated gating of postsynaptic ion channels. In the present study we investigated for behavioural endophenotypes in Kctd16-/- and Kctd16+/- mice. Compared with wild-type (WT) littermates, auditory and contextual fear conditioning were normal in both Kctd16-/- and Kctd16+/- mice. When fear memory was tested on the following day, Kctd16-/- mice exhibited less extinction of auditory fear memory relative to WT and Kctd16+/- mice, as well as more contextual fear memory relative to WT and, in particular, Kctd16+/- mice. Relative to WT, both Kctd16+/- and Kctd16-/- mice exhibited normal circadian activity. This study adds to the evidence that auxillary KCTD subunits of GABAB receptors contribute to the regulation of behaviours that could constitute endophenotypes for hyper-reactivity to aversive stimuli in neuropsychiatric disorders.
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Condicionamiento Clásico/fisiología , Endofenotipos , Memoria/fisiología , Canales de Potasio/deficiencia , Estimulación Acústica , Animales , Ritmo Circadiano/genética , Conducta Exploratoria/fisiología , Miedo/fisiología , Femenino , Genotipo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Canales de Potasio/genéticaRESUMEN
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.
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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
GABAB receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the central nervous system. Pharmacological activation of GABAB receptors regulates neurotransmission and neuronal excitability at pre- and postsynaptic sites. Electrophysiological activation of GABAB receptors in brain slices generally requires strong stimulus intensities. This raises the question as to whether behavioral stimuli are strong enough to activate GABAB receptors. Here we show that GABAB1a-/- mice, which constitutively lack presynaptic GABAB receptors at glutamatergic synapses, are impaired in their ability to acquire an operant learning task. In vivo recordings during the operant conditioning reveal a deficit in learning-dependent increases in synaptic strength at CA3-CA1 synapses. Moreover, GABAB1a-/- mice fail to synchronize neuronal activity in the CA1 area during the acquisition process. Our results support that activation of presynaptic hippocampal GABAB receptors is important for acquisition of a learning task and for learning-associated synaptic changes and network dynamics.
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Aprendizaje por Asociación/fisiología , Hipocampo/metabolismo , Receptores de GABA-B/metabolismo , Receptores Presinapticos/metabolismo , Sinapsis/metabolismo , Animales , Región CA1 Hipocampal/metabolismo , Región CA3 Hipocampal/metabolismo , Condicionamiento Operante , Potenciación a Largo Plazo , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Plasticidad Neuronal , Células Piramidales/fisiología , Potenciales SinápticosRESUMEN
GABAB receptors are crucial modulators of the behavioural effects of drug abuse, and agonists and positive allosteric modulators show promise as pharmacological strategies for anti-addiction therapeutics. GABAB receptors are functional heterodimers of GABAB1 and GABAB2 subunits. The predominant neuronal GABAB1 subunit isoforms are GABAB1a and GABAB1b. Selective ablation of these isoforms in mice revealed differential behavioural responses in fear, cognition and stress sensitivity. However, the influence of the two GABAB1 isoforms on responses to drugs of abuse is unclear. Therefore we examined the responses of GABAB1 subunit isoform null mice to cocaine in acute locomotor activity and conditioned place preference (CPP) paradigms. During habituation for the acute locomotor activity assay, GABAB1b(-/-) mice showed higher levels of locomotor activity relative to wild-type (WT) and GABAB1a(-/-) mice, in accordance with previous studies. Acute cocaine (10 mg/kg) increased locomotor activity in habituated mice of all three genotypes, with GABAB1a(-/-) mice showing sustained hyperlocomotor responses 30 min after cocaine relative to WT and GABAB1b(-/-) mice. No genotypes demonstrated a cocaine-induced place preference, however, GABAB1a(-/-) mice demonstrated enhanced locomotor sensitisation to chronic cocaine in the CPP paradigm in comparison to WT mice, whereas GABAB1b(-/-) mice failed to develop locomotor sensitisation, despite higher levels of basal locomotor activity. These findings indicate that GABAB1a and GABAB1b isoforms differentially regulate behavioural responses to cocaine, with deletion of GABAB1a enhancing cocaine-induced locomotor activity and deletion of GABAB1b protecting from cocaine-induced sensitisation.
Asunto(s)
Cocaína/administración & dosificación , Inhibidores de Captación de Dopamina/administración & dosificación , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Receptores de GABA-A/metabolismo , Animales , Condicionamiento Psicológico/efectos de los fármacos , Condicionamiento Psicológico/fisiología , Masculino , Ratones Noqueados , Isoformas de Proteínas , Receptores de GABA-A/genética , Conducta Espacial/efectos de los fármacos , Conducta Espacial/fisiologíaRESUMEN
GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-ß A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.
Asunto(s)
Proteómica/métodos , Receptores de GABA-B/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Caveolina 2/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Epítopos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G , Receptores de GABA-B/metabolismo , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Acústica/instrumentación , Ecolocación , Biología Marina/instrumentación , Ballenas/fisiología , Algoritmos , Animales , Conducta Animal , Buceo , Océano Pacífico , Espectrografía del Sonido , Transductores de PresiónRESUMEN
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.