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.
GABA(B) receptors are the G-protein-coupled receptors for γ-aminobutyric acid (GABA). KCTD8, 12, 12b, and 16 were recently identified as auxiliary GABA(B) receptor subunits and distinctly influence biophysical and pharmacological properties of the receptor response. Here we examined the expression patterns of the KCTDs in the mouse brain. Using in situ hybridization analysis, we found that most neurons express KCTD transcripts, supporting biochemical data showing that most GABA(B) receptors in the brain incorporate KCTD proteins. In the adult brain, KCTD12 and 16 have a widespread and KCTD8 and 12b a restricted expression pattern. Individual neurons can coexpress multiple KCTDs, as shown for granule cells and CA1/CA3 pyramidal cells in the hippocampus that coexpress KCTD12 and 16. In contrast, granule, Purkinje, and Golgi cells in the cerebellum selectively express one KCTD at a time. The expression levels of individual KCTD transcripts vary during postnatal brain development. Immunohistochemistry reveals that individual KCTD proteins can exhibit distinct axonal or dendritic localizations in neuronal populations. KCTDs are also detectable in nonneuronal tissues not expected to express GABA(B) receptors, suggesting that the role of KCTD proteins extends beyond GABA(B) receptors. In summary, our findings support that most brain GABA(B) receptors associate with KCTD proteins, but that the repertoire and abundance of KCTDs varies during development, among brain areas, neuronal populations, and at subcellular sites. We propose that the distinct spatial and temporal KCTD distribution patterns underlie functional differences in native GABA(B) responses.
GABA B receptors are the G-protein-coupled receptors for ␥-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. GABA B receptors are promising drug targets for a wide spectrum of psychiatric and neurological disorders. Receptor subtypes exhibit no pharmacological differences and are based on the subunit isoforms GABA B1a and GABA B1b . GABA B1a differs from GABA B1b in its ectodomain by the presence of a pair of conserved protein binding motifs, the sushi domains (SDs). Previous work showed that selectively GABA B1a contributes to heteroreceptors at glutamatergic terminals, whereas both GABA B1a and GABA B1b contribute to autoreceptors at GABAergic terminals or to postsynaptic receptors. Here, we describe GABA B1j , a secreted GABA B1 isoform comprising the two SDs. We show that the two SDs, when expressed as a soluble protein, bind to neuronal membranes with low nanomolar affinity. Soluble SD protein, when added at nanomolar concentrations to dissociated hippocampal neurons or to acute hippocampal slices, impairs the inhibitory effect of GABA B heteroreceptors on evoked and spontaneous glutamate release. In contrast, soluble SD protein neither impairs the activity of GABA B autoreceptors nor impairs the activity of postsynaptic GABA B receptors. We propose that soluble SD protein scavenges an extracellular binding partner that retains GABA B1a -containing heteroreceptors in proximity of the presynaptic release machinery. Soluble GABA B1 isoforms like GABA B1j may therefore act as dominant-negative inhibitors of heteroreceptors and control the level of GABA B -mediated inhibition at glutamatergic terminals. Of importance for drug discovery, our data also demonstrate that it is possible to selectively impair GABA B heteroreceptors by targeting their SDs.
GABA B receptors are the G-protein coupled receptors for the main inhibitory neurotransmitter in the brain, GABA. GABA B receptors were shown to associate with homo-oligomers of auxiliary KCTD8, KCTD12, KCTD12b, and KCTD16 subunits (named after their T1 K ϩ -channel tetramerization domain) that regulate G-protein signaling of the receptor. Here we provide evidence that GABA B 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 selfinteracting 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 ϩ 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 GABA B receptors and modulates physiologically induced K ϩ current responses in the hippocampus.
Background and purpose: A crucial role for the GABA B receptor in depression was proposed several years ago, but there are limited data to support this proposition. Therefore we decided to investigate the antidepressant-like activity of the selective GABA B receptor antagonists CGP 36742 and CGP 51176, and a selective agonist CGP 44532 in models of depression in rats and mice. Experimental approach: Effects of CGP 36742 and CGP 51176 as well as the agonist CGP 44532 were assessed in the forced swim test in mice. Both antagonists were also investigated in an olfactory bulbectomy (OB) model of depression in rats, while CGP 51176 was also investigated in the chronic mild stress (CMS) rat model of depression. The density of GABA B receptors in the mouse hippocampus after chronic administration of CGP 51176 was also investigated. Key results: The GABA B receptor antagonists CGP 36742 and CGP 51176 exhibited antidepressant-like activity in the forced swim test in mice. The GABA B receptor agonist CGP 44532 was not effective in this test, however, it counteracted the antidepressant-like effects of CGP 51176. The antagonists CGP 36742 and CGP 51176 were effective in an OB model of depression in rats. CGP 51176 was also effective in the CMS rat model of depression. Administration of CGP 51176 increased the density of GABA B receptors in the mouse hippocampus. Conclusions and Implications: These data suggest that selective GABA B receptor antagonists may be useful in treatment of depression, and support an important role for GABA-ergic transmission in this disorder.
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