GABAB receptors are the G protein-coupled receptors for the main inhibitory neurotransmitter in the brain, gamma-aminobutyric acid (GABA). Molecular diversity in the GABAB system arises from the GABAB1a and GABAB1b subunit isoforms that solely differ in their ectodomains by a pair of sushi repeats that is unique to GABAB1a. Using a combined genetic, physiological, and morphological approach, we now demonstrate that GABAB1 isoforms localize to distinct synaptic sites and convey separate functions in vivo. At hippocampal CA3-to-CA1 synapses, GABAB1a assembles heteroreceptors inhibiting glutamate release, while predominantly GABAB1b mediates postsynaptic inhibition. Electron microscopy reveals a synaptic distribution of GABAB1 isoforms that agrees with the observed functional differences. Transfected CA3 neurons selectively express GABAB1a in distal axons, suggesting that the sushi repeats, a conserved protein interaction motif, specify heteroreceptor localization. The constitutive absence of GABAB1a but not GABAB1b results in impaired synaptic plasticity and hippocampus-dependent memory, emphasizing molecular differences in synaptic GABAB functions.

GABA B receptors are the G-protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the brain. Two receptor subtypes, GABA B(1a,2) and GABA B(1b,2) , are formed by the assembly of GABA B1a and GABA B1b subunits with GABA B2 subunits. The GABA B1b subunit is a shorter isoform of the GABA B1a subunit lacking two N-terminal protein interaction motifs, the sushi domains. Selectively GABA B1a protein traffics into the axons of glutamatergic neurons, whereas both the GABA B1a and GABA B1b proteins traffic into the dendrites. The mechanism(s) and targeting signal(s) responsible for the selective trafficking of GABA B1a protein into axons are unknown. Here, we provide evidence that the sushi domains are axonal targeting signals that redirect GABA B1a protein from its default dendritic localization to axons. Specifically, we show that mutations in the sushi domains preventing protein interactions preclude axonal localization of GABA B1a . When fused to CD8␣, the sushi domains polarize this uniformly distributed protein to axons. Likewise, when fused to mGluR1a the sushi domains redirect this somatodendritic protein to axons, showing that the sushi domains can override dendritic targeting information in a heterologous protein. Cell surface expression of the sushi domains is not required for axonal localization of GABA B1a . Altogether, our findings are consistent with the sushi domains functioning as axonal targeting signals by interacting with axonally bound proteins along intracellular sorting pathways. Our data provide a mechanistic explanation for the selective trafficking of GABA B(1a,2) receptors into axons while at the same time identifying a well defined axonal delivery module that can be used as an experimental tool.

Synapses depend on trafficking of key membrane proteins by lateral diffusion from surface populations and by exocytosis from intracellular pools. The cell adhesion molecule neurexin (Nrxn) plays essential roles in synapses, but the dynamics and regulation of its trafficking are unknown. Here, we performed single-particle tracking and live imaging of transfected, epitope-tagged Nrxn variants in cultured rat and mouse wild-type or knock-out neurons. We observed that structurally larger ␣Nrxn molecules are more mobile in the plasma membrane than smaller ␤Nrxns because ␣Nrxns displayed higher diffusion coefficients in extrasynaptic regions and excitatory or inhibitory terminals. We found that well characterized interactions with extracellular binding partners regulate the surface mobility of Nrxns. Binding to neurexophilin-1 (Nxph1) reduced the surface diffusion of ␣Nrxns when both molecules were coexpressed. Conversely, impeding other interactions by insertion of splice sequence #4 or removal of extracellular Ca 2ϩ augmented the mobility of ␣Nrxns and ␤Nrxns. We also determined that fast axonal transport delivers Nrxns to the neuronal surface because Nrxns comigrate as cargo on synaptic vesicle protein transport vesicles (STVs). Unlike surface mobility, intracellular transport of ␤Nrxn ϩ STVs was faster than that of ␣Nrxns, but both depended on the microtubule motor protein KIF1A and neuronal activity regulated the velocity. Large spontaneous fusion of Nrxn ϩ STVs occurred simultaneously with synaptophysin on axonal membranes mostly outside of active presynaptic terminals. Surface Nrxns enriched at synaptic terminals where ␣Nrxns and Nxph1/␣Nrxns recruited GABA A R subunits. Therefore, our results identify regulated dynamic trafficking as an important property of Nrxns that corroborates their function at synapses.

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.

Organization of signalling molecules in biological membranes is crucial for cellular communication. Many receptors, ion channels and cell adhesion molecules are associated with proteins important for their trafficking, surface localization or function. These complexes are embedded in a lipid environment of varying composition. Binding affinities and stoichiometry of such complexes were so far experimentally accessible only in isolated systems or monolayers of cell culture. Visualization of molecular dynamics within signalling complexes and their correlation to specialized membrane compartments demand high temporal and spatial resolution and has been difficult to demonstrate in complex tissue like brain slices. Here we demonstrate the feasibility of single-particle tracking (SPT) in organotypic brain slices to measure molecular dynamics of lipids and transmembrane proteins in correlation to synaptic membrane compartments. This method will provide important information about the dynamics and organization of surface molecules in the complex environment of neuronal networks within brain slices.