B-type receptors for the neurotransmitter GABA (gamma-aminobutyric acid) inhibit neuronal activity through G-protein-coupled second-messenger systems, which regulate the release of neurotransmitters and the activity of ion channels and adenylyl cyclase. Physiological and biochemical studies show that there are differences in drug efficiencies at different GABA(B) receptors, so it is expected that GABA(B)-receptor (GABA(B)R) subtypes exist. Two GABA(B)-receptor splice variants have been cloned (GABA(B)R1a and GABA(B)R1b), but native GABA(B) receptors and recombinant receptors showed unexplained differences in agonist-binding potencies. Moreover, the activation of presumed effector ion channels in heterologous cells expressing the recombinant receptors proved difficult. Here we describe a new GABA(B) receptor subtype, GABA(B)R2, which does not bind available GABA(B) antagonists with measurable potency. GABA(B)R1a, GABA(B)R1b and GABA(B)R2 alone do not activate Kir3-type potassium channels efficiently, but co-expression of these receptors yields a robust coupling to activation of Kir3 channels. We provide evidence for the assembly of heteromeric GABA(B) receptors in vivo and show that GABA(B)R2 and GABA(B)R1a/b proteins immunoprecipitate and localize together at dendritic spines. The heteromeric receptor complexes exhibit a significant increase in agonist- and partial-agonist-binding potencies as compared with individual receptors and probably represent the predominant native GABA(B) receptor. Heteromeric assembly among G-protein-coupled receptors has not, to our knowledge, been described before.

AMPA-type glutamate receptors (AMPARs) are responsible for a variety of processes in the mammalian brain including fast excitatory neurotransmission, postsynaptic plasticity, or synapse development. Here, with comprehensive and quantitative proteomic analyses, we demonstrate that native AMPARs are macromolecular complexes with a large molecular diversity. This diversity results from coassembly of the known AMPAR subunits, pore-forming GluA and three types of auxiliary proteins, with 21 additional constituents, mostly secreted proteins or transmembrane proteins of different classes. Their integration at distinct abundance and stability establishes the heteromultimeric architecture of native AMPAR complexes: a defined core with a variable periphery resulting in an apparent molecular mass between 0.6 and 1 MDa. The additional constituents change the gating properties of AMPARs and provide links to the protein dynamics fundamental for the complex role of AMPARs in formation and operation of glutamatergic synapses.

Cortical synapses display remarkable structural, molecular and functional heterogeneity. Our knowledge regarding the relationship between the ultrastructural and functional parameters is still fragmented. Here we asked how the release probability and presynaptic [Ca2+] transients relate to the ultrastructure of rat hippocampal glutamatergic axon terminals. Two-photon Ca2+ imaging-derived optical quantal analysis and correlated electron microscopic reconstructions revealed a tight correlation between the release probability and the active zone area. The peak amplitude of [Ca2+] transients in single boutons also positively correlated with the active zone area. Freeze-fracture immunogold labeling revealed that the voltage-gated Ca2+ channel subunit Cav2.1 and the presynaptic protein Rim1/2 are confined to the active zone and their numbers scale linearly with the active zone area. Gold particles for Cav2.1 showed a nonrandom distribution within the active zones. Our results demonstrate that the number of several active zone proteins, including presynaptic Ca2+ channels, docked vesicles and the release probability scales linearly with the active zone area.

We studied neurogliaform neurons in the stratum lacunosum moleculare of the CA1 hippocampal area. These interneurons have short stellate dendrites and an extensive axonal arbor mainly located in the stratum lacunosum moleculare. Single-cell reverse transcription-PCR showed that these neurons were GABAergic and that the majority expressed mRNA for neuropeptide Y. Most neurogliaform neurons tested were immunoreactive for ␣-actinin-2, and many stratum lacunosum moleculare interneurons coexpressed ␣-actinin-2 and neuropeptide Y. Neurogliaform neurons received monosynaptic, DNQX-sensitive excitatory input from the perforant path, and 40 Hz stimulation of this input evoked EPSCs displaying either depression or initial facilitation, followed by depression. Paired recordings performed between neurogliaform neurons showed that 85% of pairs were electrically connected and 70% were also connected via

Glutamate receptors of the AMPA-subtype (AMPARs), together with the transmembrane AMPAR regulatory proteins (TARPs), mediate fast excitatory synaptic transmission in the mammalian brain. Here, we show by proteomic analysis that the majority of AMPARs in the rat brain are coassembled with two members of the cornichon family of transmembrane proteins, rather than with the TARPs. Coassembly with cornichon homologs 2 and 3 affects AMPARs in two ways: Cornichons increase surface expression of AMPARs, and they alter channel gating by markedly slowing deactivation and desensitization kinetics. These results demonstrate that cornichons are intrinsic auxiliary subunits of native AMPARs and provide previously unknown molecular determinants for glutamatergic neurotransmission in the central nervous system.