TY - JOUR AB - Chemical labeling of proteins with synthetic molecular probes offers the possibility to probe the functions of proteins of interest in living cells. However, the methods for covalently labeling targeted proteins using complementary peptide tag-probe pairs are still limited, irrespective of the versatility of such pairs in biological research. Herein, we report the new CysHis tag-Ni(II) probe pair for the specific covalent labeling of proteins. A broad-range evaluation of the reactivity profiles of the probe and the CysHis peptide tag afforded a tag-probe pair with an optimized and high labeling selectivity and reactivity. In particular, the labeling specificity of this pair was notably improved compared to the previously reported one. This pair was successfully utilized for the fluorescence imaging of membrane proteins on the surfaces of living cells, demonstrating its potential utility in biological research. AU - Zenmyo, Naoki AU - Tokumaru, Hiroki AU - Uchinomiya, Shohei AU - Fuchida, Hirokazu AU - Tabata, Shigekazu AU - Hamachi, Itaru AU - Shigemoto, Ryuichi AU - Ojida, Akio ID - 6659 IS - 5 JF - Bulletin of the Chemical Society of Japan SN - 00092673 TI - Optimized reaction pair of the CysHis tag and Ni(II)-NTA probe for highly selective chemical labeling of membrane proteins VL - 92 ER - TY - JOUR AB - Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels control electrical rhythmicity and excitability in the heart and brain, but the function of HCN channels at the subcellular level in axons remains poorly understood. Here, we show that the action potential conduction velocity in both myelinated and unmyelinated central axons can be bidirectionally modulated by a HCN channel blocker, cyclic adenosine monophosphate (cAMP), and neuromodulators. Recordings from mouse cerebellar mossy fiber boutons show that HCN channels ensure reliable high-frequency firing and are strongly modulated by cAMP (EC50 40 mM; estimated endogenous cAMP concentration 13 mM). In addition, immunogold-electron microscopy revealed HCN2 as the dominating subunit in cerebellar mossy fibers. Computational modeling indicated that HCN2 channels control conduction velocity primarily by altering the resting membrane potential and are associated with significant metabolic costs. These results suggest that the cAMP-HCN pathway provides neuromodulators with an opportunity to finely tune energy consumption and temporal delays across axons in the brain. AU - Byczkowicz, Niklas AU - Eshra, Abdelmoneim AU - Montanaro-Punzengruber, Jacqueline-Claire AU - Trevisiol, Andrea AU - Hirrlinger, Johannes AU - Kole, Maarten Hp AU - Shigemoto, Ryuichi AU - Hallermann, Stefan ID - 6868 JF - eLife TI - HCN channel-mediated neuromodulation can control action potential velocity and fidelity in central axons VL - 8 ER - TY - JOUR AU - Kasugai, Yu AU - Vogel, Elisabeth AU - Hörtnagl, Heide AU - Schönherr, Sabine AU - Paradiso, Enrica AU - Hauschild, Markus AU - Göbel, Georg AU - Milenkovic, Ivan AU - Peterschmitt, Yvan AU - Tasan, Ramon AU - Sperk, Günther AU - Shigemoto, Ryuichi AU - Sieghart, Werner AU - Singewald, Nicolas AU - Lüthi, Andreas AU - Ferraguti, Francesco ID - 7099 IS - 4 JF - Neuron SN - 0896-6273 TI - Structural and functional remodeling of amygdala GABAergic synapses in associative fear learning VL - 104 ER - TY - JOUR AB - 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. AU - Klotz, Lisa AU - Wendler, Olaf AU - Frischknecht, Renato AU - Shigemoto, Ryuichi AU - Schulze, Holger AU - Enz, Ralf ID - 7179 IS - 12 JF - FASEB Journal TI - Localization of group II and III metabotropic glutamate receptors at pre- and postsynaptic sites of inner hair cell ribbon synapses VL - 33 ER - TY - JOUR AB - 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. AU - Erdem, Fatma Asli AU - Ilic, Marija AU - Koppensteiner, Peter AU - Gołacki, Jakub AU - Lubec, Gert AU - Freissmuth, Michael AU - Sandtner, Walter ID - 7398 IS - 8 JF - The Journal of General Physiology SN - 0022-1295 TI - A comparison of the transport kinetics of glycine transporter 1 and glycine transporter 2 VL - 151 ER -