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• W2026872153 abstract "Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain. Glutamate receptors play major roles in excitatory transmission in the vertebrate brain. Among ionotropic glutamate receptors (AMPA, kainate, NMDA), AMPA receptors mediate fast synaptic transmission and require TARP auxiliary subunits. NMDA receptors and kainate receptors play roles in synaptic transmission, but it remains uncertain whether these ionotropic glutamate receptors also have essential subunits. Using a proteomic screen, we have identified NETO2, a brain-specific protein of unknown function, as an interactor with kainate-type glutamate receptors. NETO2 modulates the channel properties of recombinant and native kainate receptors without affecting trafficking of the receptors and also modulates kainate-receptor-mediated mEPSCs. Furthermore, we found that kainate receptors regulate the surface expression of NETO2 and that NETO2 protein levels and surface expression are decreased in mice lacking the kainate receptor GluR6. The results show that NETO2 is a kainate receptor subunit with significant effects on glutamate signaling mechanisms in brain. Excitatory synaptic transmission in brain is primarily mediated by the neurotransmitter glutamate. Glutamate released from presynaptic terminals binds to three classes of ionotropic glutamate receptors, which are pharmacologically classified as AMPA- (amino-3-hydroxy-5-methylisoxazole-4-propionic acid), NMDA- (N-methyl-D-aspartic acid), and kainate-sensitive glutamate receptors (Dingledine et al., 1999Dingledine R. Borges K. Bowie D. Traynelis S.F. The glutamate receptor ion channels.Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar, Hollmann and Heinemann, 1994Hollmann M. Heinemann S. Cloned glutamate receptors.Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3585) Google Scholar, Seeburg, 1993Seeburg P.H. The Trends Neurosci./TiPS Lecture. The molecular biology of mammalian glutamate receptor channels.Trends Neurosci. 1993; 16: 359-365Abstract Full Text PDF PubMed Scopus (802) Google Scholar). AMPA receptors mediate fast synaptic transmission, whereas NMDA receptors are involved in synaptic plasticity. Kainate receptors play multiple roles in synaptic transmission. Postsynaptic kainate receptors mediate slow EPSCs (Castillo et al., 1997Castillo P.E. Malenka R.C. Nicoll R.A. Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons.Nature. 1997; 388: 182-186Crossref PubMed Scopus (420) Google Scholar, Kidd and Isaac, 1999Kidd F.L. Isaac J.T. Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses.Nature. 1999; 400: 569-573Crossref PubMed Scopus (186) Google Scholar, Vignes and Collingridge, 1997Vignes M. Collingridge G.L. The synaptic activation of kainate receptors.Nature. 1997; 388: 179-182Crossref PubMed Scopus (354) Google Scholar), and presynaptic kainate receptors modulate the release of the excitatory and inhibitory neurotransmitters glutamate and GABA (Chittajallu et al., 1996Chittajallu R. Vignes M. Dev K.K. Barnes J.M. Collingridge G.L. Henley J.M. Regulation of glutamate release by presynaptic kainate receptors in the hippocampus.Nature. 1996; 379: 78-81Crossref PubMed Scopus (340) Google Scholar, Clarke et al., 1997Clarke V.R. Ballyk B.A. Hoo K.H. Mandelzys A. Pellizzari A. Bath C.P. Thomas J. Sharpe E.F. Davies C.H. Ornstein P.L. et al.A hippocampal GluR5 kainate receptor regulating inhibitory synaptic transmission.Nature. 1997; 389: 599-603Crossref PubMed Scopus (379) Google Scholar, Kamiya and Ozawa, 1998Kamiya H. Ozawa S. Kainate receptor-mediated inhibition of presynaptic Ca2+ influx and EPSP in area CA1 of the rat hippocampus.J. Physiol. 1998; 509: 833-845Crossref PubMed Scopus (114) Google Scholar, Rodriguez-Moreno et al., 1997Rodriguez-Moreno A. Herreras O. Lerma J. Kainate receptors presynaptically downregulate GABAergic inhibition in the rat hippocampus.Neuron. 1997; 19: 893-901Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Furthermore, presynaptic kainate receptors are involved in long-term potentiation at mossy fiber-CA3 pyramidal cells in hippocampus (Bortolotto et al., 1999Bortolotto Z.A. Clarke V.R. Delany C.M. Parry M.C. Smolders I. Vignes M. Ho K.H. Miu P. Brinton B.T. Fantaske R. et al.Kainate receptors are involved in synaptic plasticity.Nature. 1999; 402: 297-301Crossref PubMed Scopus (265) Google Scholar, Contractor et al., 2001Contractor A. Swanson G. Heinemann S.F. Kainate receptors are involved in short- and long-term plasticity at mossy fiber synapses in the hippocampus.Neuron. 2001; 29: 209-216Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, Schmitz et al., 2001Schmitz D. Mellor J. Nicoll R.A. Presynaptic kainate receptor mediation of frequency facilitation at hippocampal mossy fiber synapses.Science. 2001; 291: 1972-1976Crossref PubMed Scopus (225) Google Scholar). Although mice in which kainate receptor expression is genetically disrupted show no kainate receptor activity, overexpression of kainate receptors does not enhance kainate-receptor-mediated excitatory postsynaptic current (EPSC), suggesting that native kainate receptors may contain additional modulatory proteins. For example, faithful reconstitution of native AMPA receptor properties in heterologous cells requires coexpression of transmembrane AMPA receptor auxiliary subunits (TARPs) (Nicoll et al., 2006Nicoll R.A. Tomita S. Bredt D.S. Auxiliary subunits assist AMPA-type glutamate receptors.Science. 2006; 311: 1253-1256Crossref PubMed Scopus (272) Google Scholar). Several cytoplasmic proteins (SAP90/PSD95, the cadherin/catenin complex, KRIP6, Actinfillin, PICK1, Syntenin, GRIP) have been identified as kainate receptor interactors (Coussen et al., 2002Coussen F. Normand E. Marchal C. Costet P. Choquet D. Lambert M. Mege R.M. Mulle C. Recruitment of the kainate receptor subunit glutamate receptor 6 by cadherin/catenin complexes.J. Neurosci. 2002; 22: 6426-6436Crossref PubMed Google Scholar, Garcia et al., 1998Garcia E.P. Mehta S. Blair L.A. Wells D.G. Shang J. Fukushima T. Fallon J.R. Garner C.C. Marshall J. SAP90 binds and clusters kainate receptors causing incomplete desensitization.Neuron. 1998; 21: 727-739Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, Hirbec et al., 2003Hirbec H. Francis J.C. Lauri S.E. Braithwaite S.P. Coussen F. Mulle C. Dev K.K. Coutinho V. Meyer G. Isaac J.T. et al.Rapid and differential regulation of AMPA and kainate receptors at hippocampal mossy fibre synapses by PICK1 and GRIP.Neuron. 2003; 37: 625-638Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, Laezza et al., 2007Laezza F. Wilding T.J. Sequeira S. Coussen F. Zhang X.Z. Hill-Robinson R. Mulle C. Huettner J.E. Craig A.M. KRIP6: a novel BTB/kelch protein regulating function of kainate receptors.Mol. Cell. Neurosci. 2007; 34: 539-550Crossref PubMed Scopus (36) Google Scholar, Salinas et al., 2006Salinas G.D. Blair L.A. Needleman L.A. Gonzales J.D. Chen Y. Li M. Singer J.D. Marshall J. Actinfilin is a Cul3 substrate adaptor, linking GluR6 kainate receptor subunits to the ubiquitin-proteasome pathway.J. Biol. Chem. 2006; 281: 40164-40173Crossref PubMed Scopus (61) Google Scholar). The primary effect of these proteins is to modulate receptor localization. Garcia et al., 1998Garcia E.P. Mehta S. Blair L.A. Wells D.G. Shang J. Fukushima T. Fallon J.R. Garner C.C. Marshall J. SAP90 binds and clusters kainate receptors causing incomplete desensitization.Neuron. 1998; 21: 727-739Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar showed with whole-cell recording that SAP90/PSD95 causes incomplete receptor desensitization in heterologous cells. However, subsequent studies at greater time resolutions using outside-out patch membranes showed that SAP90/PSD95 does not modulate the rate at which receptors desensitize, but rather accelerates recovery from desensitization (Bowie et al., 2003Bowie D. Garcia E.P. Marshall J. Traynelis S.F. Lange G.D. Allosteric regulation and spatial distribution of kainate receptors bound to ancillary proteins.J. Physiol. 2003; 547: 373-385Crossref PubMed Scopus (54) Google Scholar). Recently, KRIP6 was also shown to modulate receptor kinetics. KRIP6 enhanced the ratio of steady-state to peak currents, but did not significantly alter decay kinetics in heterologous cells (Laezza et al., 2007Laezza F. Wilding T.J. Sequeira S. Coussen F. Zhang X.Z. Hill-Robinson R. Mulle C. Huettner J.E. Craig A.M. KRIP6: a novel BTB/kelch protein regulating function of kainate receptors.Mol. Cell. Neurosci. 2007; 34: 539-550Crossref PubMed Scopus (36) Google Scholar). Importantly, enhancement of kainate-receptor-mediated EPSCs by overexpression of these interactors has not yet been shown. In this study, we identified a brain-specific transmembrane protein of unknown function, NETO2, using a proteomic screen. NETO2 slows the decay kinetics of kainate receptors in heterologous cells without affecting receptor expression at the cell surface. Single-channel analysis showed that NETO2 also increases the open probability (Popen) of kainate-receptor channels, resulting in significantly larger peak glutamate-evoked currents. NETO2 modulated the agonist sensitivity of kainate receptors in heterologous cells and neurons. Importantly, NETO2 slowed the decay of kainate-receptor-mediated EPSCs, demonstrating that it directly influences synaptic transmission. The total amount of NETO2 is decreased in mice lacking the kainate receptor subunit GluR6, and kainate receptors increase the surface expression of NETO2 in both heterologous cells and neurons. The results indicate that NETO2 is an accessory subunit of neuronal kainate receptors that has important effects on receptor function. The kainate receptor subunit GluR6 plays major roles in kainate receptor function (Mulle et al., 1998Mulle C. Sailer A. Perez-Otano I. Dickinson-Anson H. Castillo P.E. Bureau I. Maron C. Gage F.H. Mann J.R. Bettler B. Heinemann S.F. Altered synaptic physiology and reduced susceptibility to kainate-induced seizures in GluR6-deficient mice.Nature. 1998; 392: 601-605Crossref PubMed Scopus (378) Google Scholar) and is highly expressed in cerebellum (Bahn et al., 1994Bahn S. Volk B. Wisden W. Kainate receptor gene expression in the developing rat brain.J. Neurosci. 1994; 14: 5525-5547Crossref PubMed Google Scholar, Smith et al., 1999Smith T.C. Wang L.Y. Howe J.R. Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells.J. Physiol. 1999; 517: 51-58Crossref PubMed Scopus (38) Google Scholar). To identify proteins that interact with kainate receptors, we used rat cerebella for coimmunoprecipitation experiments with anti GluR6/7 antibody, followed by silver staining. Mass spectrometry analysis of bands immunoprecipitated with anti GluR6/7 antibody identified a rat ortholog of NETO2/Btcl2 (99% shared identity of amino acids), a brain-specific mouse protein of unknown function (Michishita et al., 2004Michishita M. Ikeda T. Nakashiba T. Ogawa M. Tashiro K. Honjo T. Doi K. Itohara S. Endo S. Expression of Btcl2, a novel member of Btcl gene family, during development of the central nervous system.Brain Res. Dev. Brain Res. 2004; 153: 135-142Crossref PubMed Scopus (17) Google Scholar, Stohr et al., 2002Stohr H. Berger C. Frohlich S. Weber B.H. A novel gene encoding a putative transmembrane protein with two extracellular CUB domains and a low-density lipoprotein class A module: isolation of alternatively spliced isoforms in retina and brain.Gene. 2002; 286: 223-231Crossref PubMed Scopus (53) Google Scholar), as well as AKAP8, the kainate receptor subunits GluR6 and GluR7, and a major contaminant, keratin (Figure 1A and Table S1 available online). NETO2 and GluR6/7 were also coimmunoprecipitated with an anti GluR5 antibody that also recognizes GluR6 directly (Figure S1A available online), whereas AKAP8 was not detected (Figure S1B), suggesting that the anti GluR6/7 antibody may directly recognize AKAP8. To test this possibility, we used Cos-7 cells transfected with FLAG-AKAP8 for immunoprecipitation experiments with anti GluR6/7 antibody. We found that AKAP8 was weakly immunoprecipitated with anti GluR6/7 antibody in the absence of GluR6/7 expression (Figure S1C). We therefore concluded that NETO2, but not AKAP8, is a kainate receptor interactor. To determine if NETO2 interacted specifically with kainate receptors, we generated antibodies against the cytoplasmic domain of NETO2. NETO2 is a 525 amino acid protein that contains a signal peptide, and the expected molecular weight of NETO2 protein after cleavage of the signal peptide is 56 kDa. Anti NETO2 antibody recognized 58–60 kDa bands in NETO2 transfected CHO cells, but not untransfected cells, and similar bands were recognized in lysates from rat brain and mouse cerebellar granule cell cultures (Figure S2). Thus, we concluded that the anti NETO2 antibody recognized NETO2 in brain and transfected cells. We used anti NETO2 antibody for coimmunoprecipitation experiments on rat cerebellar lysates. We found that NETO2 interacts specifically with kainate receptors, but not with AMPA and NMDA receptors (Figure 1B). In contrast, TARPs interact specifically with AMPA receptors, but not with kainate and NMDA receptors (Figure 1B). NETO2 codistributed with the kainate receptor subunits GluR6/7 and KA2, and was highly enriched in the postsynaptic density (PSD) fraction, along with PSD95 and the NMDA receptor subunit NR1 (Figure 1C). Sequence analysis indicates that NETO2 contains one transmembrane domain, as well as two CUB domains and one LDLa domain that are extracellular. Like the NMDA receptor subunit NR1, NETO2 contains EndoH sensitive sugars (2–3 kDa), consistent with the idea that NETO2 is a transmembrane protein (Figure 1D). The predicted structure of NETO2 is substantially different from that of the TARP family of AMPA receptor auxiliary subunits (Figure 1E). Here NETO2 is identified as a CUB-domain-containing protein that binds to ion channels in the vertebrate. In Caenorhabditis elegans, CUB-domain-containing proteins LEV-10 and SOL-1 were identified as modulators of acetylcholine receptors and GLR-1 AMPA receptors (Gally et al., 2004Gally C. Eimer S. Richmond J.E. Bessereau J.L. A transmembrane protein required for acetylcholine receptor clustering in Caenorhabditis elegans.Nature. 2004; 431: 578-582Crossref PubMed Scopus (105) Google Scholar, Zheng et al., 2004Zheng Y. Mellem J.E. Brockie P.J. Madsen D.M. Maricq A.V. SOL-1 is a CUB-domain protein required for GLR-1 glutamate receptor function in C. elegans.Nature. 2004; 427: 451-457Crossref PubMed Scopus (102) Google Scholar, Zheng et al., 2006Zheng Y. Brockie P.J. Mellem J.E. Madsen D.M. Walker C.S. Francis M.M. Maricq A.V. SOL-1 is an auxiliary subunit that modulates the gating of GLR-1 glutamate receptors in Caenorhabditis elegans.Proc. Natl. Acad. Sci. USA. 2006; 103: 1100-1105Crossref PubMed Scopus (31) Google Scholar). Interestingly, the domain structure of NETO2 is more similar to the domain structure of invertebrate proteins that modulate acetylcholine receptors (LEV-10) than SOL-1, which modulates GLR-1 AMPA receptors (Figure S3). In addition, NETO2 is similar to the hypothetical proteins Q9XUU2 in C. elegans and Q9VYC7 in Drosophila melanogaster (Figure S3 and Discussion). To begin to explore the functional role of NETO2 interactions with kainate receptors, we coexpressed these proteins in Xenopus laevis oocytes, in which protein expression could be more tightly regulated by complementary RNA (cRNA) injection (in comparison with transfection of cDNAs in mammalian cells). The detection of glutamate-evoked currents in oocytes injected with 0.5 ng GluR6 cRNA by two-electrode voltage-clamp recording (TEVC) required reduction of desensitization with concanavalin A (Wong and Mayer, 1993Wong L.A. Mayer M.L. Differential modulation by cyclothiazide and concanavalin A of desensitization at native alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-preferring glutamate receptors.Mol. Pharmacol. 1993; 44: 504-510PubMed Google Scholar). In contrast, large glutamate-evoked currents were routinely recorded without concanavalin A treatment in oocytes coinjected with 0.5 ng NETO2 cRNA and 0.5 ng GluR6 cRNA, whereas coexpression of NETO2 had no effect on the activity of AMPA receptors or stargazin-like TARP/AMPA receptor complexes expressed at the minimum levels to detect further enhancement (Figures 2A, 2B, and S4). To determine whether the NETO2-associated increase in GluR6 currents was due to an increase in receptor expression at the cell surface, we measured in parallel glutamate-evoked currents by TEVC and the surface expression of kainate receptors by chemiluminescence. Oocytes were injected with extracellular HA-epitope-tagged GluR6 at nonsaturating amounts (0.5 ng) of HA-GluR6 cRNA (Figure S5) with varying amounts of NETO2 cRNA (Tomita et al., 2005Tomita S. Adesnik H. Sekiguchi M. Zhang W. Wada K. Howe J.R. Nicoll R.A. Bredt D.S. Stargazin modulates AMPA receptor gating and trafficking by distinct domains.Nature. 2005; 435: 1052-1058Crossref PubMed Scopus (380) Google Scholar). We found that NETO2 enhanced maximal glutamate-evoked currents in a dose-dependent manner, but had no effect on surface expression (Figure 2C and the raw data in Figure S6), suggesting that NETO2 enhances GluR6 currents by modulating the functional properties of the receptors, perhaps by slowing desensitization. To examine NETO2 modulation of receptor properties at a better time resolution, we expressed NETO2 and GluR6 in tsA201 cells and applied glutamate to outside-out patches from the transfected cells with a fast piezoelectric system. Coexpression of GluR6 and NETO2 at a cDNA ratio of 1:10 resulted in significant slowing of both desensitization and deactivation (Figures 3A–3C). Relative to the corresponding values for GluR6 alone, the NETO2-associated increase in mean weighted tau values was 500% for desensitization and 64% for deactivation. The results obtained with cDNA ratios of 1:10 and 1:30 were similar, indicating that most GluR6 receptors in the patches studied contained NETO2. In addition, NETO2 did not modulate desensitization of GluR1 AMPA receptors (time constant of desensitization: GluR1 alone, 2.4 ± 0.2 ms, n = 5; GluR1 + NETO2, 2.2 ± 0.5 ms, n = 4). Two-pulse protocols showed that NETO2 also causes GluR6 receptors to recover faster from desensitization, especially at short interpulse intervals (Figure 3D). Together, the slower entry into desensitization and faster recovery seen with NETO2 coexpression resulted in a 9-fold enhancement of steady-state currents (0.11 ± 0.02% versus 1.01% ± 0.24% of the peak current, n = 6 and 9, respectively). Notably, the difference in kinetics suggests that there is no endogenous NETO2 in tsA201 cells, because human and rat NETO2 should have similar effects on receptor kinetics given the high degree of identity (97%) the two proteins share. To determine the effect of NETO2 on unitary receptor properties, we made concentration jumps on patches containing only a few channels (Figure 3E). As reported before (Swanson et al., 1996Swanson G.T. Feldmeyer D. Kaneda M. Cull-Candy S.G. Effect of RNA editing and subunit co-assembly single-channel properties of recombinant kainate receptors.J. Physiol. 1996; 492: 129-142Crossref PubMed Scopus (152) Google Scholar), GluR6-Q channels displayed three open levels with conductances of approximately 7, 17, and 26 pS. NETO2 coexpression had no effect on unitary conductance and did not alter the relative frequency or the mean duration of openings to each conductance level (Table 1). However, the duration of bursts of openings (tcrit = 4 ms) was clearly longer with NETO2 (Figures 3E and 3F) and the mean duration of these bursts was increased significantly (Table 1).Table 1Effect of NETO2 on the Unitary Properties of GluR6-Q ChannelsGluR6-QGluR6-Q + NETO2Conductance (pS)O17.4 ± 0.3 (24.7%)7.4 ± 0.2 (18.2%)O216.9 ± 0.4 (43.2%)16.4 ± 0.5 (51.3%)O327.0 ± 0.8 (32.1%)25.5 ± 0.6 (30.5%)Open time (ms)O10.87 ± 0.130.70 ± 0.05O20.60 ± 0.090.80 ± 0.07O30.81 ± 0.140.89 ± 0.07Burst length (ms)0.64 ± 0.080.66 ± 0.104.60 ± 0.387.45 ± 0.82∗Mean ± SEM values from four or five patches are listed. Conductance levels and open times for each level were estimated with QuB software. Bursts were defined as a series of openings (to any level) that were separated by shuttings briefer than 4 ms. The distributions of burst durations were fitted with two exponential components. The fast component was similar in all distributions examined. ∗p < 0.05. Open table in a new tab Mean ± SEM values from four or five patches are listed. Conductance levels and open times for each level were estimated with QuB software. Bursts were defined as a series of openings (to any level) that were separated by shuttings briefer than 4 ms. The distributions of burst durations were fitted with two exponential components. The fast component was similar in all distributions examined. ∗p < 0.05. It was evident from inspection of the records that NETO2 coexpression dramatically increased channel activity. In patches in which the maximum number of receptors that open simultaneously was estimated to be three or four, the number of jumps that produced no detectable openings (“failures”) was much higher in the absence of NETO2 coexpression (Figure 3E). Our measurements of the relative frequency of openings to each conductance level with and without NETO2 (Table 1) were used to calculate the mean single-channel current, which together with the number of active receptors in the patch allowed us to estimate the Popen at the peak of the ensemble current (peak Popen). NETO2 coexpression increased peak Popen significantly (Figure 3G). In total, the results show that the inclusion of NETO2 in kainate receptor assemblies modulates both the amplitude and kinetics of ensemble currents evoked by rapid pulses of glutamate, leading to a marked increase in charge transfer. Our results suggest that the mechanism underlying NETO2-mediated kainate receptor modulation differs from TARP modulation of AMPA receptors. TARPs slow both the deactivation and desensitization of AMPA receptors to similar extents, suggesting that they decrease the activation energy for channel opening, whereas NETO2 has larger effects on desensitization and may primarily alter the rate constants governing transits in and out of desensitized states (Figure 3H). We next examined whether NETO2 modulates the properties of the synaptic receptors that mediate kainate-receptor miniature EPSCs (mEPSCs). We used cerebellar granule cells for these studies because their small size allowed us to measure EPSC kinetics accurately. To minimize the contribution of AMPA receptors, we used neurons from stargazer mice, which lack AMPA-receptor-mediated EPSCs (Chen et al., 2000Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms.Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar, Hashimoto et al., 1999Hashimoto K. Fukaya M. Qiao X. Sakimura K. Watanabe M. Kano M. Impairment of AMPA receptor function in cerebellar granule cells of ataxic mutant mouse stargazer.J. Neurosci. 1999; 19: 6027-6036Crossref PubMed Google Scholar). As published previously (Chen et al., 2000Chen L. Chetkovich D.M. Petralia R.S. Sweeney N.T. Kawasaki Y. Wenthold R.J. Bredt D.S. Nicoll R.A. Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms.Nature. 2000; 408: 936-943Crossref PubMed Scopus (9) Google Scholar, Cho et al., 2007Cho C.H. St-Gelais F. Zhang W. Tomita S. Howe J.R. Two Families of TARP Isoforms that Have Distinct Effects on the Kinetic Properties of AMPA Receptors and Synaptic Currents.Neuron. 2007; 55: 890-904Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, Milstein et al., 2007Milstein A.D. Zhou W. Karimzadegan S. Bredt D.S. Nicoll R.A. TARP Subtypes Differentially and Dose-Dependently Control Synaptic AMPA Receptor Gating.Neuron. 2007; 55: 905-918Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), we observed AMPA-receptor-mediated mEPSCs in cerebellar granule cells from wild-type mice, but not in cells from stargazer mice (data not shown). NMDA receptors, GABA receptors, and voltage-gated sodium channels were blocked by the inclusion of AP-5, picrotoxin, and tetrodotoxin (respectively) in the external solution. We did not detect mEPSCs in stargazer granule cells, even after transfecting the neurons with GluR6, although we routinely detected AMPA receptor mEPSCs in neurons in parallel cultures transfected with stargazin. We hypothesized that the low peak Popen of GluR6 channels limited our detection of kainate receptors at synapses. We therefore transfected granule cells with a GluR6 mutant in which substitution of lysine for arginine at position 696 reduces receptor desensitization (Priel et al., 2006Priel A. Selak S. Lerma J. Stern-Bach Y. Block of kainate receptor desensitization uncovers a key trafficking checkpoint.Neuron. 2006; 52: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In oocytes injected with cRNAs, NETO2 enhanced glutamate-evoked currents of GluR6 (K696R) receptors without altering surface expression of the receptors (Figure S7). We did observe mEPSCs in some neurons transfected with GluR6 (K696R) alone, although the frequency of the events was very low (Figure 4A). In contrast, mEPSCs were observed in most neurons cotransfected with NETO2 and GluR6 (K696R), and the frequency of these events was significantly greater (Figure 4A). Examples of representative mEPSCs are shown in Figures 4B and 4C. The mEPSCs detected in each type of transfected neuron were mediated by kainate receptors, as confirmed by their resistance to block by selective AMPA receptor antagonists (50 μM GYKI 53655 and 100 μM SYM 2206) (Figures 4B and 4C). The decay kinetics of the kainate-receptor-mediated mEPSCs with and without NETO2 coexpression were different and were slower in neurons cotransfected with NETO2 (Figure 4D), and NETO2 significantly increased the half-width of individual mEPSCs, as well as the decay time and charge transfer of ensemble averages from individual neurons (Figure 4E). Additionally, in cerebellar granule cells from stargazer mice, NETO2 slowed the decay of spontaneous kainate-receptor EPSCs in neurons transfected with GluR5. The time constants obtained from fitting average EPSCs were 0.87 ± 0.10 ms for neurons transfected with GluR5 alone and 2.6 ± 0.36 ms for neurons cotransfected with GluR5 and NETO2 (p < 0.05). The results indicate that NETO2 can modulate kainate-receptor-mediated synaptic transmission. It is well known that kainate-receptor-mediated EPSCs are less than 10% of the amplitude of AMPA-receptor-mediated EPSCs (Castillo et al., 1997Castillo P.E. Malenka R.C. Nicoll R.A. Kainate receptors mediate a slow postsynaptic current in hippocampal CA3 neurons.Nature. 1997; 388: 182-186Crossref PubMed Scopus (420) Google Scholar, Kidd and Isaac, 1999Kidd F.L. Isaac J.T. Developmental and activity-dependent regulation of kainate receptors at thalamocortical synapses.Nature. 1999; 400: 569-573Crossref PubMed Scopus (186) Google Scholar, Vignes and Collingridge, 1997Vignes M. Collingridge G.L. The synaptic activation of kainate receptors.Nature. 1997; 388: 179-182Crossref PubMed Scopus (354) Google Scholar), and in cerebellar granule cells whole-cell kainate receptor currents are on average smaller than 20 pA even when desensitization is reduced with concanavalin A (Pemberton et al., 1998Pemberton K.E. Belcher S.M. Ripellino J.A. Howe J.R. High-affinity kainate-type ion channels in rat cerebellar granule cells.J. Physiol. 1998; 510: 401-420Crossref PubMed Scopus (48) Google Scholar, Smith et al., 1999Smith T.C. Wang L.Y. Howe J.R. Distinct kainate receptor phenotypes in immature and mature mouse cerebellar granule cells.J. Physiol. 1999; 517: 51-58Crossref PubMed Scopus (38) Google Scholar). Because we were unable to routinely detect native kainate receptor mEPSCs in granule cells (or hippocampal neurons), we used a pharmacological approach to determine whether endogenous kainate receptors contain NETO2. For AMPA receptors, modulation of receptor kinetics by TARP auxiliary subunits is associated with changes in the relative efficacy of glutamate and kainate (Tomita et al., 2005Tomita S. Adesnik H. Sekiguchi M. Zhang W. Wada K. Howe J.R. Nicoll R.A. Bredt D.S. Stargazin modulates AMPA receptor gating and trafficking by distinct domains.Nature. 2005; 435: 1052-1058Crossref PubMed Scopus (380) Google Scholar). To determin" @default.
• W2026872153 created "2016-06-24" @default.
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• W2026872153 date "2009-02-01" @default.
• W2026872153 modified "2023-10-16" @default.
• W2026872153 title "A Transmembrane Accessory Subunit that Modulates Kainate-Type Glutamate Receptors" @default.
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