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 GABA, the main inhibitory neurotransmitter in the brain. GABA B receptors are abundant on dendritic spines, where they dampen postsynaptic excitability and inhibit Ca 2+ 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 GABA B -mediated inhibition. We found that NMDA application to cultured hippocampal neurons promotes dynamindependent endocytosis of GABA B receptors. NMDA-dependent internalization of GABA B receptors requires activation of Ca 2+ /Calmodulindependent protein kinase II (CaMKII), which associates with GABA B receptors in vivo and phosphorylates serine 867 (S867) in the intracellular C terminus of the GABA B1 subunit. Blockade of either CaMKII or phosphorylation of S867 renders GABA B receptors refractory to NMDA-mediated internalization. Time-lapse two-photon imaging of organotypic hippocampal slices reveals that activation of NMDA receptors removes GABA B receptors within minutes from the surface of dendritic spines and shafts. NMDA-dependent S867 phosphorylation and internalization is predominantly detectable with the GABA B1b 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 GABA B receptors to activate K + channels. Thus, our data support that NMDA receptor activity endocytoses postsynaptic GABA B receptors through CaMKIImediated phosphorylation of S867. This provides a means to spare NMDA receptors at individual glutamatergic synapses from reciprocal inhibition through GABA B receptors.γ-aminobutyric acid | spines | trafficking | synaptic plasticity | GABAB

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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.

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