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 Gβγ 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 Gβγ 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 Gβγ 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 Gβγ at the receptor relies on the interaction of KCTD proteins with a loop protruding from the seven-bladed propeller of Gβ subunits. The binding site is shared between Gβ1 and Gβ2, limiting the interaction of KCTD proteins to these particular Gβ isoforms. Substituting residues in the KCTD binding site of Gβ1 with those from Gβ3 hinders the preassembly of Gβγ with GBRs, delays onset and prolongs rise time of receptor-activated K+ currents. The KCTD-Gβ interface, therefore, represents a target for pharmacological modulation of channel gating by GBRs.

• MeSH
• draslíkové kanály metabolismus   genetika   MeSH
• gating iontového kanálu * fyziologie   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• proteiny vázající GTP - beta-podjednotky * metabolismus   genetika   MeSH
• proteiny vázající GTP - gama-podjednotky * metabolismus   genetika   MeSH
• receptory GABA-B * metabolismus   genetika   MeSH
• Xenopus laevis MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

High-frequency oscillations (HFOs) represent an electrographic biomarker of endogenous epileptogenicity and seizure-generating tissue that proved clinically useful in presurgical planning and delineating the resection area. In the neocortex, the clinical observations on HFOs are not sufficiently supported by experimental studies stemming from a lack of realistic neocortical epilepsy models that could provide an explanation of the pathophysiological substrates of neocortical HFOs. In this study, we explored pathological epileptiform network phenomena, particularly HFOs, in a highly realistic murine model of neocortical epilepsy due to focal cortical dysplasia (FCD) type II. FCD was induced in mice by the expression of the human pathogenic mTOR gene mutation during embryonic stages of brain development. Electrographic recordings from multiple cortical regions in freely moving animals with FCD and epilepsy demonstrated that the FCD lesion generates HFOs from all frequency ranges, i.e., gamma, ripples, and fast ripples up to 800 Hz. Gamma-ripples were recorded almost exclusively in FCD animals, while fast ripples occurred in controls as well, although at a lower rate. Gamma-ripple activity is particularly valuable for localizing the FCD lesion, surpassing the utility of fast ripples that were also observed in control animals, although at significantly lower rates. Propagating HFOs occurred outside the FCD, and the contralateral cortex also generated HFOs independently of the FCD, pointing to a wider FCD network dysfunction. Optogenetic activation of neurons carrying mTOR mutation and expressing Channelrhodopsin-2 evoked fast ripple oscillations that displayed spectral and morphological profiles analogous to spontaneous oscillations. This study brings experimental evidence that FCD type II generates pathological HFOs across all frequency bands and provides information about the spatiotemporal properties of each HFO subtype in FCD. The study shows that mutated neurons represent a functionally interconnected and active component of the FCD network, as they can induce interictal epileptiform phenomena and HFOs.

• MeSH
• elektroencefalografie MeSH
• epilepsie * MeSH
• fokální kortikální dysplazie * MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• TOR serin-threoninkinasy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• buněčná membrána MeSH
• GABA MeSH
• hluchota * MeSH
• kognice MeSH
• receptory GABA-B * MeSH
• savci MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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.

• MeSH
• glukokortikoidy * MeSH
• kortikosteron * MeSH
• lidé MeSH
• myši MeSH
• receptory GABA-A genetika   metabolismus   MeSH
• receptory GABA metabolismus   MeSH
• systém hypofýza - nadledviny metabolismus   MeSH
• systém hypotalamus-hypofýza metabolismus   MeSH
• úzkost MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Vysokoškolská učebnice pro studující biomedicíncké techniky a informatiky, která se zaměřuje na biochemii.

• MeSH
• biochemie MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• Učební osnovy. Vyučovací předměty. Učebnice
• NLK Obory
• biochemie
• biomedicínské inženýrství
• NLK Publikační typ
• učebnice vysokých škol

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.

• MeSH
• ablace * MeSH
• agonisté receptorů GABA-A farmakologie   MeSH
• corpus trapezoideum fyziologie   MeSH
• inhibiční postsynaptické potenciály fyziologie   MeSH
• kochlea patofyziologie   chirurgie   MeSH
• krysa rodu rattus MeSH
• nervový přenos * MeSH
• nervový útlum účinky léků   fyziologie   MeSH
• novorozená zvířata MeSH
• receptory GABA-A fyziologie   MeSH
• receptory glycinu agonisté   metabolismus   fyziologie   MeSH
• sluchové kmenové evokované potenciály fyziologie   MeSH
• transportéry VIAAT metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• draslíkové kanály genetika   metabolismus   MeSH
• elektrofyziologické jevy genetika   MeSH
• excitační postsynaptické potenciály genetika   MeSH
• kinetika MeSH
• křečci praví MeSH
• metoda terčíkového zámku MeSH
• mozek - chemie genetika   MeSH
• myši knockoutované MeSH
• myši MeSH
• receptory GABA-B genetika   metabolismus   MeSH
• receptory KIR metabolismus   MeSH
• receptory spřažené s G-proteiny metabolismus   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mechanisms controlling the metabotropic γ-aminobutyric acid receptor (GABAB) cell surface stability are still poorly understood. In contrast with many other G protein-coupled receptors (GPCR), it is not subject to agonist-promoted internalization, but is constitutively internalized and rapidly down-regulated. In search of novel interacting proteins regulating receptor fate, we report that the ubiquitin-specific protease 14 (USP14) interacts with the GABAB(1b)subunit's second intracellular loop. Probing the receptor for ubiquitination using bioluminescence resonance energy transfer (BRET), we detected a constitutive and phorbol 12-myristate 13-acetate (PMA)-induced ubiquitination of the receptor at the cell surface. PMA also increased internalization and accelerated receptor degradation. Overexpression of USP14 decreased ubiquitination while treatment with a small molecule inhibitor of the deubiquitinase (IU1) increased receptor ubiquitination. Treatment with the internalization inhibitor Dynasore blunted both USP14 and IU1 effects on the receptor ubiquitination state, suggesting a post-endocytic site of action. Overexpression of USP14 also led to an accelerated degradation of GABABin a catalytically independent fashion. We thus propose a model whereby cell surface ubiquitination precedes endocytosis, after which USP14 acts as an ubiquitin-binding protein that targets the ubiquitinated receptor to lysosomal degradation and promotes its deubiquitination.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• endocytóza účinky léků   MeSH
• HEK293 buňky MeSH
• hydrazony farmakologie   MeSH
• lidé MeSH
• luciferasy genetika   metabolismus   MeSH
• luminescentní proteiny genetika   metabolismus   MeSH
• lyzozomy metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• posttranslační úpravy proteinů * MeSH
• proteinkinasa C genetika   metabolismus   MeSH
• proteolýza MeSH
• receptory GABA-B genetika   metabolismus   MeSH
• reportérové geny MeSH
• sekvence aminokyselin MeSH
• signální transdukce MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• thiolesterasa ubikvitinu genetika   metabolismus   MeSH
• ubikvitin genetika   metabolismus   MeSH
• ubikvitinace MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• GABA metabolismus   MeSH
• glycin metabolismus   MeSH
• krysa rodu rattus MeSH
• mozkový kmen cytologie   MeSH
• neparametrická statistika MeSH
• neurony cytologie   MeSH
• potkani Wistar MeSH
• presynaptická zakončení metabolismus   ultrastruktura   MeSH
• receptory glycinu metabolismus   ultrastruktura   MeSH
• synapse metabolismus   ultrastruktura   MeSH
• techniky in vitro MeSH
• vezikulární transportní protein 1 pro glutamát metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH