GABAB receptors : physiological functions and mechanisms of diversity

GABA(B) receptors are the G-protein-coupled receptors (GPCRs) for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the central nervous system. GABA(B) receptors are implicated in the etiology of a variety of psychiatric disorders and are considered attractive drug targets. With the cloning of GABA(B) receptor subunits 13 years ago, substantial progress was made in the understanding of the molecular structure, physiology, and pharmacology of these receptors. However, it remained puzzling that native studies demonstrated a heterogeneity of GABA(B) responses that contrasted with a very limited diversity of cloned GABA(B) receptor subunits. Until recently, the only firmly established molecular diversity consisted of two GABA(B1) subunit isoforms, GABA(B1a) and GABA(B1b), which assemble with GABA(B2) subunits to generate heterodimeric GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Using genetic, ultrastructural, biochemical, and electrophysiological approaches, it has been possible to identify functional properties that segregate with these two receptors. Moreover, receptor modifications and factors that can alter the receptor response have been identified. Most importantly, recent data reveal the existence of a family of auxiliary GABA(B) receptor subunits that assemble as tetramers with the C-terminal domain of GABA(B2) subunits and drastically alter pharmacology and kinetics of the receptor response. The data are most consistent with native GABA(B) receptors minimally forming dimeric assemblies of units composed of GABA(B1), GABA(B2), and a tetramer of auxiliary subunits. This represents a substantial departure from current structural concepts for GPCRs

GABA B receptors modulate Ca2+ but not G protein‐gated inwardly rectifying K+ channels in cerebrospinal‐fluid contacting neurones of mouse brainstem

International audienceMedullo-spinal CSF contacting neurones (CSF-cNs) located around the central canal are conserved in all vertebrates and suggested to be a novel sensory system intrinsic to the CNS. CSF-cNs receive GABAergic inhibitory synaptic inputs involving ionotropic GABAA receptors, but the contribution of metabotropic GABAB receptors (GABAB -Rs) has not yet been studied. Here, we indicate that CSF-cNs express functional GABAB -Rs that inhibit postsynaptic calcium channels but fail to activate inhibitory potassium channel of the Kir3-type. We further show that GABAB -Rs localise presynaptically on GABAergic and glutamatergic synaptic inputs contacting CSF-cNs, where they inhibit the release of GABA and glutamate. Our data are the first to address the function of GABAB -Rs in CSF-cNs and show that on the presynaptic side they exert a classical synaptic modulation whereas at the postsynaptic level they have an atypical action by modulating calcium signalling without inducing potassium-dependent inhibition

Synapse loss in cortex of agrin-deficient mice after genetic rescue of perinatal death

Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex. Most importantly, we examined the brains of agrin-deficient mice whose perinatal death was prevented by the selective expression of agrin in motor neurons. We find that the number of presynaptic and postsynaptic specializations is strongly reduced in the cortex of 5- to 7-week-old mice. Consistent with a reduction in the number of synapses, the frequency of miniature postsynaptic currents was greatly decreased. In accordance with the synaptic localization of agrin to excitatory synapses, changes in the frequency were only detected for excitatory but not inhibitory synapses. Moreover, we find that the muscle-specific receptor tyrosine kinase MuSK, which is known to be an essential component of agrin-induced signaling at the neuromuscular junction, is also localized to a subset of excitatory synapses. Finally, some components of the mitogen-activated protein (MAP) kinase pathway, which has been shown to be activated by agrin in cultured neurons, are deregulated in agrin-deficient mice. In summary, our results provide strong evidence that agrin plays an important role in the formation and/or the maintenance of excitatory synapses in the brain, and we provide evidence that this function involves MAP kinase signaling

The GABAB1a isoform mediates heterosynaptic depression at hippocampal mossy fiber synapses

GABA(B) receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which associate with GABA(B2) subunits to form pharmacologically indistinguishable GABA(B(1a,2)) and GABA(B(1b,2)) receptors. Studies with mice selectively expressing GABA(B1a) or GABA(B1b) subunits revealed that GABA(B(1a,2)) receptors are more abundant than GABA(B(1b,2)) receptors at glutamatergic terminals. Accordingly, it was found that GABA(B(1a,2)) receptors are more efficient than GABA(B(1b,2)) receptors in inhibiting glutamate release when maximally activated by exogenous application of the agonist baclofen. Here, we used a combination of genetic, ultrastructural and electrophysiological approaches to analyze to what extent GABA(B(1a,2)) and GABA(B(1b,2)) receptors inhibit glutamate release in response to physiological activation. We first show that at hippocampal mossy fiber (MF)-CA3 pyramidal neuron synapses more GABA(B1a) than GABA(B1b) protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations <or=1 microm, both GABA(B(1a,2)) and GABA(B(1b,2)) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABA(B(1a,2)) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABA(B(1a,2)) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABA(B(1a,2)) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABA(B) receptors. Our data demonstrate that the difference in GABA(B1a) and GABA(B1b) protein levels at MF terminals is sufficient to produce a strictly GABA(B1a)-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABA(B1a) and GABA(B1b) proteins is of regulatory relevance

A single polycystic kidney disease 2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem.

International audienceCerebrospinal fluid contacting neurons (CSF-cNs) are found around the central canal of all vertebrates. They present a typical morphology, with a single dendrite that projects into the cavity and ends in the CSF with a protuberance. These anatomical features have led to the suggestion that CSF-cNs might have sensory functions, either by sensing CSF movement or composition, but the physiological mechanisms for any such role are unknown. This hypothesis was recently supported by the demonstration that in several vertebrate species medullo-spinal CSF-cNs selectively express Polycystic Kidney Disease 2-Like 1 proteins (PKD2L1). PKD2L1 are members of the 'transient receptor potential (TRP)' superfamily, form non-selective cationic channels of high conductance, are regulated by various stimuli including protons and are therefore suggested to act as sensory receptors. Using patch-clamp whole-cell recordings of CSF-cNs in brainstem slices obtained from wild type and mutant PKD2L1 mice, we demonstrate that spontaneously active unitary currents in CSF-cNs are due to PKD2L1 channels that are capable, with a single opening, of triggering action potentials. Thus PKD2L1 might contribute to the setting of CSF-cN spiking activity. We also reveal that CSF-cNs have the capacity of discriminating between alkalinization and acidification following activation of specific conductances (PKD2L1 vs. ASIC) generating specific responses. Altogether, this study reinforces the idea that CSF-cNs represent sensory neurons intrinsic to the central nervous system and suggests a role for PKD2L1 channels as spike generators.Copyright © 2015 Elsevier Ltd. All rights reserved

A mouse model for visualization of GABA(B) receptors

GABA(B) receptors are the G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes are based on the subunit isoforms GABA(B1a) and GABA(B1b), which combine with GABA(B2) subunits to form heteromeric receptors. Here, we used a modified bacterial artificial chromosome (BAC) containing the GABA(B1) gene to generate transgenic mice expressing GABA(B1a) and GABA(B1b) subunits fused to the enhanced green fluorescence protein (eGFP). We demonstrate that the GABA(B1)-eGFP fusion proteins reproduce the cellular expression patterns of endogenous GABA(B1) proteins in the brain and in peripheral tissue. Crossing the GABA(B1)-eGFP BAC transgene into the GABA(B1) (-/-) background restores pre and postsynaptic GABA(B) functions, showing that the GABA(B1)-eGFP fusion proteins substitute for the lack of endogenous GABA(B1) proteins. Finally, we demonstrate that the GABA(B1)-eGFP fusion proteins replicate the temporal expression patterns of native GABA(B) receptors in cultured neurons. These transgenic mice therefore provide a validated tool for direct visualization of native GABA(B) receptors

NMDA receptor-dependent GABAB receptor internalization via CaMKII phosphorylation of serine 867 in GABAB1

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

Glial Endozepines Reverse High-Fat Diet-Induced Obesity by Enhancing Hypothalamic Response to Peripheral Leptin

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Opposite effects of KCTD subunit domains on GABA(B) receptor-mediated desensitization

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

Native GABA(B) receptors are heteromultimers with a family of auxiliary subunits

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