@article{7179, abstract = {Glutamate is the major excitatory neurotransmitter in the CNS binding to a variety of glutamate receptors. Metabotropic glutamate receptors (mGluR1 to mGluR8) can act excitatory or inhibitory, depending on associated signal cascades. Expression and localization of inhibitory acting mGluRs at inner hair cells (IHCs) in the cochlea are largely unknown. Here, we analyzed expression of mGluR2, mGluR3, mGluR4, mGluR6, mGluR7, and mGluR8 and investigated their localization with respect to the presynaptic ribbon of IHC synapses. We detected transcripts for mGluR2, mGluR3, and mGluR4 as well as for mGluR7a, mGluR7b, mGluR8a, and mGluR8b splice variants. Using receptor-specific antibodies in cochlear wholemounts, we found expression of mGluR2, mGluR4, and mGluR8b close to presynaptic ribbons. Super resolution and confocal microscopy in combination with 3-dimensional reconstructions indicated a postsynaptic localization of mGluR2 that overlaps with postsynaptic density protein 95 on dendrites of afferent type I spiral ganglion neurons. In contrast, mGluR4 and mGluR8b were expressed at the presynapse close to IHC ribbons. In summary, we localized in detail 3 mGluR types at IHC ribbon synapses, providing a fundament for new therapeutical strategies that could protect the cochlea against noxious stimuli and excitotoxicity.}, author = {Klotz, Lisa and Wendler, Olaf and Frischknecht, Renato and Shigemoto, Ryuichi and Schulze, Holger and Enz, Ralf}, issn = {15306860}, journal = {FASEB Journal}, number = {12}, pages = {13734--13746}, publisher = {FASEB}, title = {{Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses}}, doi = {10.1096/fj.201901543R}, volume = {33}, year = {2019}, } @article{7398, abstract = {Transporters of the solute carrier 6 (SLC6) family translocate their cognate substrate together with Na+ and Cl−. Detailed kinetic models exist for the transporters of GABA (GAT1/SLC6A1) and the monoamines dopamine (DAT/SLC6A3) and serotonin (SERT/SLC6A4). Here, we posited that the transport cycle of individual SLC6 transporters reflects the physiological requirements they operate under. We tested this hypothesis by analyzing the transport cycle of glycine transporter 1 (GlyT1/SLC6A9) and glycine transporter 2 (GlyT2/SLC6A5). GlyT2 is the only SLC6 family member known to translocate glycine, Na+, and Cl− in a 1:3:1 stoichiometry. We analyzed partial reactions in real time by electrophysiological recordings. Contrary to monoamine transporters, both GlyTs were found to have a high transport capacity driven by rapid return of the empty transporter after release of Cl− on the intracellular side. Rapid cycling of both GlyTs was further supported by highly cooperative binding of cosubstrate ions and substrate such that their forward transport mode was maintained even under conditions of elevated intracellular Na+ or Cl−. The most important differences in the transport cycle of GlyT1 and GlyT2 arose from the kinetics of charge movement and the resulting voltage-dependent rate-limiting reactions: the kinetics of GlyT1 were governed by transition of the substrate-bound transporter from outward- to inward-facing conformations, whereas the kinetics of GlyT2 were governed by Na+ binding (or a related conformational change). Kinetic modeling showed that the kinetics of GlyT1 are ideally suited for supplying the extracellular glycine levels required for NMDA receptor activation.}, author = {Erdem, Fatma Asli and Ilic, Marija and Koppensteiner, Peter and Gołacki, Jakub and Lubec, Gert and Freissmuth, Michael and Sandtner, Walter}, issn = {1540-7748}, journal = {The Journal of General Physiology}, number = {8}, pages = {1035--1050}, publisher = {Rockefeller University Press}, title = {{A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2}}, doi = {10.1085/jgp.201912318}, volume = {151}, year = {2019}, } @article{7391, abstract = {Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.}, author = {Tabata, Shigekazu and Jevtic, Marijo and Kurashige, Nobutaka and Fuchida, Hirokazu and Kido, Munetsugu and Tani, Kazushi and Zenmyo, Naoki and Uchinomiya, Shohei and Harada, Harumi and Itakura, Makoto and Hamachi, Itaru and Shigemoto, Ryuichi and Ojida, Akio}, issn = {2589-0042}, journal = {iScience}, number = {12}, pages = {256--268}, publisher = {Elsevier}, title = {{Electron microscopic detection of single membrane proteins by a specific chemical labeling}}, doi = {10.1016/j.isci.2019.11.025}, volume = {22}, year = {2019}, } @inbook{562, abstract = {Primary neuronal cell culture preparations are widely used to investigate synaptic functions. This chapter describes a detailed protocol for the preparation of a neuronal cell culture in which giant calyx-type synaptic terminals are formed. This chapter also presents detailed protocols for utilizing the main technical advantages provided by such a preparation, namely, labeling and imaging of synaptic organelles and electrophysiological recordings directly from presynaptic terminals.}, author = {Dimitrov, Dimitar and Guillaud, Laurent and Eguchi, Kohgaku and Takahashi, Tomoyuki}, booktitle = {Neurotrophic Factors}, editor = {Skaper, Stephen D.}, pages = {201 -- 215}, publisher = {Springer}, title = {{Culture of mouse giant central nervous system synapses and application for imaging and electrophysiological analyses}}, doi = {10.1007/978-1-4939-7571-6_15}, volume = {1727}, year = {2018}, } @article{41, abstract = {The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.}, author = {Luján, Rafæl and Aguado, Carolina and Ciruela, Francisco and Arus, Xavier and Martín Belmonte, Alejandro and Alfaro Ruiz, Rocío and Martinez Gomez, Jesus and De La Ossa, Luis and Watanabe, Masahiko and Adelman, John and Shigemoto, Ryuichi and Fukazawa, Yugo}, issn = {16625102}, journal = {Frontiers in Cellular Neuroscience}, publisher = {Frontiers Media}, title = {{Sk2 channels associate with mGlu1α receptors and CaV2.1 channels in Purkinje cells}}, doi = {10.3389/fncel.2018.00311}, volume = {12}, year = {2018}, }