Akos Kulik

Functional proteomics identify cornichon proteins as auxiliary subunits of AMPA receptors.

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.

Native GABAB receptors are heteromultimers with a family of auxiliary subunits

GABAB receptors are the G-protein-coupled receptors for γ-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 (Cav) and inward-rectifier potassium (Kir) channels. Molecular cloning revealed that functional GABAB receptors are formed by the heteromeric assembly of GABAB1 with GABAB2 subunits. However, cloned GABAB(1,2) receptors failed to reproduce the functional diversity observed with native GABAB receptors. Here we show by functional proteomics that GABAB receptors in the brain are high-molecular-mass complexes of GABAB1, GABAB2 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 GABAB2 as tetramers. This co-assembly changes the properties of the GABAB(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 GABAB receptors that determine the pharmacology and kinetics of the receptor response.

Release probability of hippocampal glutamatergic terminals scales with the size of the active zone

Cortical synapses have structural, molecular and functional heterogeneity; our knowledge regarding the relationship between their ultrastructural and functional parameters is still fragmented. Here we asked how the neurotransmitter 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. 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 calcium 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 labeling Cav2.1 were nonrandomly distributed in the active zones. Our results demonstrate that the numbers of several active-zone proteins, including presynaptic calcium channels, as well as the number of docked vesicles and the release probability, scale linearly with the active-zone area.

High-Resolution Proteomics Unravel Architecture and Molecular Diversity of Native AMPA Receptor Complexes

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.

Nanodomain Coupling between Ca2+ Channels and Ca2+ Sensors Promotes Fast and Efficient Transmitter Release at a Cortical GABAergic Synapse

It is generally thought that transmitter release at mammalian central synapses is triggered by Ca2+ microdomains, implying loose coupling between presynaptic Ca2+ channels and Ca2+ sensors of exocytosis. Here we show that Ca2+ channel subunit immunoreactivity is highly concentrated in the active zone of GABAergic presynaptic terminals of putative parvalbumin-containing basket cells in the hippocampus. Paired recording combined with presynaptic patch pipette perfusion revealed that GABA release at basket cell-granule cell synapses is sensitive to millimolar concentrations of the fast Ca2+ chelator BAPTA but insensitive to the slow Ca2+ chelator EGTA. These results show that Ca2+ source and Ca2+ sensor are tightly coupled at this synapse, with distances in the range of 10-20 nm. Models of Ca2+ inflow-exocytosis coupling further reveal that the tightness of coupling increases efficacy, speed, and temporal precision of transmitter release. Thus, tight coupling contributes to fast feedforward and feedback inhibition in the hippocampal network.

Neurogliaform Neurons Form a Novel Inhibitory Network in the Hippocampal CA1 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 α-actinin-2, and many stratum lacunosum moleculare interneurons coexpressed α-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 GABAergic synapses. Injection of sine waveforms into neurons during paired recordings resulted in transmission of the waveforms through the electrical synapse. Unitary IPSCs recorded from neurogliaform pairs readily fatigued, had a slow decay, and had a strong depression of the synaptic response at a 5 Hz stimulation frequency that was antagonized by the GABAB antagonist (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid (CGP55845). The amplitude of the first IPSC during the 5 Hz stimulation was also increased by CGP55845, suggesting a tonic inhibition of synaptic transmission. A small unitary GABAB-mediated IPSC could also be detected, providing the first evidence for such a component between GABAergic interneurons. Electron microscopic localization of the GABAB1 subunit at neurogliaform synapses revealed the protein in both presynaptic and postsynaptic membranes. Our data disclose a novel interneuronal network well suited for modulating the flow of information between the entorhinal cortex and CA1 hippocampus.

Distinct localization of GABAB receptors relative to synaptic sites in the rat cerebellum and ventrobasal thalamus

Metabotropic γ‐aminobutyric acid receptors (GABABRs) are involved in modulation of synaptic transmission and activity of cerebellar and thalamic neurons. We used subtype‐specific antibodies in pre‐ and postembedding immunohistochemistry combined with three‐dimensional reconstruction of labelled profiles and quantification of immunoparticles to reveal the subcellular distribution of pre‐ and postsynaptic GABABR1a/b and GABABR2 in the rat cerebellum and ventrobasal thalamus. GABABR1a/b and R2 were extensively colocalized in most brain regions including the cerebellum and thalamus. In the cerebellum, immunoreactivity for both subtypes was prevalent in the molecular layer. The most intense immunoreactivity was found in Purkinje cell spines with a high density of immunoparticles at extrasynaptic sites peaking at around 240 nm from glutamatergic synapses between spines and parallel fibre varicosities. This is in contrast to dendrites at sites around GABAergic synapses where sparse and random distribution was found for both subtypes. In addition, more than one‐tenth of the synaptic membrane specialization of spine–parallel fibre synapses were labelled at pre‐ or postsynaptic sites. Weak immunolabelling for both subtypes was also seen in parallel fibres but only rarely in GABAergic axons. In the ventrobasal thalamus, immunolabelling for both receptor subtypes was intense over the dendritic field of thalamocortical cells. Electron microscopy demonstrated an extrasynaptic localization of GABABR1a/b and R2 exclusively in postsynaptic elements. Quantitative analysis further revealed the density of GABABR1a/b around GABAergic synapses was higher than glutamatergic synapses on thalamocortical cell dendrites. The distinct localization of GABABRs relative to synaptic sites in the cerebellum and ventrobasal thalamus suggests that GABABRs differentially regulate activity of different neuronal populations.

Compartment-Dependent Colocalization of Kir3.2-Containing K+ Channels and GABAB Receptors in Hippocampal Pyramidal Cells

G-protein-coupled inwardly rectifying K+ channels (Kir3 channels) coupled to metabotropic GABAB receptors are essential for the control of neuronal excitation. To determine the distribution of Kir3 channels and their spatial relationship to GABAB receptors on hippocampal pyramidal cells, we used a high-resolution immunocytochemical approach. Immunoreactivity for the Kir3.2 subunit was most abundant postsynaptically and localized to the extrasynaptic plasma membrane of dendritic shafts and spines of principal cells. Quantitative analysis of immunogold particles for Kir3.2 revealed an enrichment of the protein around putative glutamatergic synapses on dendritic spines, similar to that of GABAB1. Consistent with this observation, a high degree of coclustering of Kir3.2 and GABAB1 was revealed around excitatory synapses by the highly sensitive SDS-digested freeze–fracture replica immunolabeling. In contrast, in dendritic shafts receptors and channels were found to be mainly segregated. These results suggest that Kir3.2-containing K+ channels on dendritic spines preferentially mediate the effect of GABA, whereas channels on dendritic shafts are likely to be activated by other neurotransmitters as well. Thus, Kir3 channels, localized to different subcellular compartments of hippocampal principal cells, appear to be differentially involved in synaptic integration in pyramidal cell dendrites.

Immunocytochemical localization of the α1A subunit of the P/Q-type calcium channel in the rat cerebellum

Among various types of low‐ and high‐threshold calcium channels, the high voltage‐activated P/Q‐type channel is the most abundant in the cerebellum. These P/Q‐type channels are involved in the regulation of neurotransmitter release and in the integration of dendritic inputs. We used an antibody specific for the α1A subunit of the P/Q‐type channel in quantitative pre‐embedding immunogold labelling combined with three‐dimensional reconstruction to reveal the subcellular distribution of pre‐ and postsynaptic P/Q‐type channels in the rat cerebellum. At the light microscopic level, immunoreactivity for the α1A protein was prevalent in the molecular layer, whereas immunostaining was moderate in the somata of Purkinje cells and weak in the granule cell layer. At the electron microscopic level, the most intense immunoreactivity for the α1A subunit was found in the presynaptic active zone of parallel fibre varicosities. The dendritic spines of Purkinje cells were also strongly labelled with the highest density of immunoparticles detected within 180 nm from the edge of the asymmetrical parallel fibre–Purkinje cell synapses. By contrast, the immunolabelling was sparse in climbing fibre varicosities and axon terminals of GABAergic cells, and weak and diffuse in dendritic shafts of Purkinje cells. The association of the α1A subunit with the glutamatergic parallel fibre–Purkinje cell synapses suggests that presynaptic channels have a major role in the mediation of excitatory neurotransmission, whereas postsynaptic channels are likely to be involved in depolarization‐induced generation of local calcium transients in Purkinje cells.

The GABAB1a Isoform Mediates Heterosynaptic Depression at Hippocampal Mossy Fiber Synapses

GABAB receptor subtypes are based on the subunit isoforms GABAB1a and GABAB1b, which associate with GABAB2 subunits to form pharmacologically indistinguishable GABAB(1a,2) and GABAB(1b,2) receptors. Studies with mice selectively expressing GABAB1a or GABAB1b subunits revealed that GABAB(1a,2) receptors are more abundant than GABAB(1b,2) receptors at glutamatergic terminals. Accordingly, it was found that GABAB(1a,2) receptors are more efficient than GABAB(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 GABAB(1a,2) and GABAB(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 GABAB1a than GABAB1b protein is present at presynaptic sites, consistent with the findings at other glutamatergic synapses. In the presence of baclofen at concentrations ≥1 μm, both GABAB(1a,2) and GABAB(1b,2) receptors contribute to presynaptic inhibition of glutamate release. However, at lower concentrations of baclofen, selectively GABAB(1a,2) receptors contribute to presynaptic inhibition. Remarkably, exclusively GABAB(1a,2) receptors inhibit glutamate release in response to synaptically released GABA. Specifically, we demonstrate that selectively GABAB(1a,2) receptors mediate heterosynaptic depression of MF transmission, a physiological phenomenon involving transsynaptic inhibition of glutamate release via presynaptic GABAB receptors. Our data demonstrate that the difference in GABAB1a and GABAB1b protein levels at MF terminals is sufficient to produce a strictly GABAB1a-specific effect under physiological conditions. This consolidates that the differential subcellular localization of the GABAB1a and GABAB1b proteins is of regulatory relevance.