Abstract
It has been shown that subunit composition is the main determinant of the synaptic or extrasynaptic localization of GABAA receptors (GABAARs). Synaptic and extrasynaptic GABAARs are involved in phasic and tonic inhibition, respectively. It has been proposed that synaptic GABAARs bind to the postsynaptic gephyrin/collybistin (Geph/CB) lattice, but not the typically extrasynaptic GABAARs. Nevertheless, there are no studies of the direct binding of various types of GABAARs with the submembranous Geph/CB lattice in the absence of other synaptic proteins, some of which are known to interact with GABAARs. We have reconstituted GABAARs of various subunit compositions, together with the Geph/CB scaffold, in HEK293 cells, and have investigated the recruitment of surface GABAARs by submembranous Geph/CB clusters. Results show that the typically synaptic α1β3γ2 GABAARs were trapped by submembranous Geph/CB clusters. The α5β3γ2 GABAARs, which are both synaptic and extrasynaptic, were also trapped by Geph/CB clusters. Extrasynaptic α4β3δ GABAARs consistently showed little or no trapping by the Geph/CB clusters. However, the extrasynaptic α6β3δ, α1β3, α6β3 (and less α4β3) GABAARs were highly trapped by the Geph/CB clusters. AMPA and NMDA glutamate receptors were not trapped. The results suggest: (I) in the absence of other synaptic molecules, the Geph/CB lattice has the capacity to trap not only synaptic but also several typically extrasynaptic GABAARs; (II) the Geph/CB lattice is important but does not play a decisive role in the synaptic localization of GABAARs; and (III) in neurons there must be mechanisms preventing the trapping of several typically extrasynaptic GABAARs by the postsynaptic Geph/CB lattice.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Availability of Data and Materials
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Change history
24 February 2021
A Correction to this paper has been published: https://doi.org/10.1007/s10571-021-01061-y
References
Alldred MJ, Mulder-Rosi J, Lingenfelter SE, Chen G, Luscher B (2005) Distinct gamma2 subunit domains mediate clustering and synaptic function of postsynaptic GABAA receptors and gephyrin. J Neurosci 25:594–603. https://doi.org/10.1523/JNEUROSCI.4011-04.2005
Alvarez FJ (2017) Gephyrin and the regulation of synaptic strength and dynamics at glycinergic inhibitory synapses. Brain Res Bull 129:50–65. https://doi.org/10.1016/j.brainresbull.2016.09.003
Angelotti TP, Macdonald RL (1993) Assembly of GABAA receptor subunits: alpha 1 beta 1 and alpha 1 beta 1 gamma 2S subunits produce unique ion channels with dissimilar single-channel properties. J Neurosci 13:1429–1440
Angelotti TP, Uhler MD, Macdonald RL (1993) Assembly of GABAA receptor subunits: analysis of transient single-cell expression utilizing a fluorescent substrate/marker gene technique. J Neurosci 13:1418–1428
Arslan A, von Engelhardt J, Wisden W (2014) Cytoplasmic domain of delta subunit is important for the extra-synaptic targeting of GABAA receptor subtypes. J Integr Neurosci 13:617–631. https://doi.org/10.1142/S0219635214500228
Barberis A (2019) Postsynaptic plasticity of GABAergic synapses. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2019.05.020
Bencsits E, Ebert V, Tretter V, Sieghart W (1999) A significant part of native gamma-aminobutyric AcidA receptors containing alpha4 subunits do not contain gamma or delta subunits. J Biol Chem 274:19613–19616
Boileau AJ, Pearce RA, Czajkowski C (2010) The short splice variant of the gamma 2 subunit acts as an external modulator of GABA(A) receptor function. J Neurosci 30:4895–4903. https://doi.org/10.1523/JNEUROSCI.5039-09.2010
Botzolakis EJ, Gurba KN, Lagrange AH, Feng HJ, Stanic AK, Hu N, Macdonald RL (2016) Comparison of gamma-aminobutyric acid, type A (GABAA), receptor alphabetagamma and alphabetadelta expression using flow cytometry and electrophysiology: evidence for alternative subunit stoichiometries and arrangements. J Biol Chem 291:20440–20461. https://doi.org/10.1074/jbc.M115.698860
Brady ML, Jacob TC (2015) Synaptic localization of alpha5 GABA (A) receptors via gephyrin interaction regulates dendritic outgrowth and spine maturation. Dev Neurobiol 75:1241–1251. https://doi.org/10.1002/dneu.22280
Brickley SG, Mody I (2012) Extrasynaptic GABA(A) receptors: their function in the CNS and implications for disease. Neuron 73:23–34. https://doi.org/10.1016/j.neuron.2011.12.012
Brickley SG, Cull-Candy SG, Farrant M (1999) Single-channel properties of synaptic and extrasynaptic GABAA receptors suggest differential targeting of receptor subtypes. J Neurosci 19:2960–2973. https://doi.org/10.1523/JNEUROSCI.19-08-02960.1999
Brunig I, Scotti E, Sidler C, Fritschy JM (2002) Intact sorting, targeting, and clustering of gamma-aminobutyric acid A receptor subtypes in hippocampal neurons in vitro. J Comp Neurol 443:43–55. https://doi.org/10.1002/cne.10102
Campbell BFN, Tyagarajan SK (2019) Cellular mechanisms contributing to the functional heterogeneity of GABAergic synapses. Front Mol Neurosci 12:187. https://doi.org/10.3389/fnmol.2019.00187
Chandra D et al (2006) GABAA receptor alpha 4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol. Proc Natl Acad Sci USA 103:15230–15235. https://doi.org/10.1073/pnas.0604304103
Charych EI, Yu W, Miralles CP, Serwanski DR, Li X, Rubio M, De Blas AL (2004a) The brefeldin A-inhibited GDP/GTP exchange factor 2, a protein involved in vesicular trafficking, interacts with the beta subunits of the GABA receptors. J Neurochem 90:173–189. https://doi.org/10.1111/j.1471-4159.2004.02481.x
Charych EI et al (2004b) A four PDZ domain-containing splice variant form of GRIP1 is localized in GABAergic and glutamatergic synapses in the brain. J Biol Chem 279:38978–38990. https://doi.org/10.1074/jbc.M405786200
Chiou TT et al (2011) Differential regulation of the postsynaptic clustering of gamma-aminobutyric acid type A (GABAA) receptors by collybistin isoforms. J Biol Chem 286:22456–22468. https://doi.org/10.1074/jbc.M111.236190
Choii G, Ko J (2015) Gephyrin: a central GABAergic synapse organizer. Exp Mol Med 47:e158. https://doi.org/10.1038/emm.2015.5
Choquet D, Triller A (2013) The dynamic synapse. Neuron 80:691–703. https://doi.org/10.1016/j.neuron.2013.10.013
Christie SB, de Blas AL (2002) alpha5 Subunit-containing GABA(A) receptors form clusters at GABAergic synapses in hippocampal cultures. Neuroreport 13:2355–2358. https://doi.org/10.1097/01.wnr.0000045008.30898.dd
Christie SB et al (2002a) Synaptic and extrasynaptic GABAA receptor and gephyrin clusters. Prog Brain Res 136:157–180. https://doi.org/10.1016/S0079-6123(02)36015-1
Christie SB, Miralles CP, De Blas AL (2002b) GABAergic innervation organizes synaptic and extrasynaptic GABAA receptor clustering in cultured hippocampal neurons. J Neurosci 22:684–697. https://doi.org/10.1523/JNEUROSCI.22-03-00684.2002
Christie SB, Li RW, Miralles CP, Yang B, De Blas AL (2006) Clustered and non-clustered GABAA receptors in cultured hippocampal neurons. Mol Cell Neurosci 31:1–14. https://doi.org/10.1016/j.mcn.2005.08.014
Chuang SH, Reddy DS (2018) Genetic and molecular regulation of extrasynaptic GABA-A receptors in the brain: therapeutic insights for epilepsy. J Pharmacol Exp Ther 364:180–197. https://doi.org/10.1124/jpet.117.244673
Connolly CN, Wooltorton JR, Smart TG, Moss SJ (1996a) Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits. Proc Natl Acad Sci USA 93:9899–9904. https://doi.org/10.1073/pnas.93.18.9899
Connolly CN, Krishek BJ, McDonald BJ, Smart TG, Moss SJ (1996b) Assembly and cell surface expression of heteromeric and homomeric gamma-aminobutyric acid type A receptors. J Biol Chem 271:89–96. https://doi.org/10.1074/jbc.271.1.89
Connolly CN, Uren JM, Thomas P, Gorrie GH, Gibson A, Smart TG, Moss SJ (1999a) Subcellular localization and endocytosis of homomeric gamma2 subunit splice variants of gamma-aminobutyric acid type A receptors. Mol Cell Neurosci 13:259–271. https://doi.org/10.1006/mcne.1999.0746
Connolly CN, Kittler JT, Thomas P, Uren JM, Brandon NJ, Smart TG, Moss SJ (1999b) Cell surface stability of gamma-aminobutyric acid type A receptors. Dependence on protein kinase C activity and subunit composition. J Biol Chem 274:36565–36572. https://doi.org/10.1074/jbc.274.51.36565
Crestani F et al (2002) Trace fear conditioning involves hippocampal alpha5 GABA(A) receptors. Proc Natl Acad Sci USA 99:8980–8985. https://doi.org/10.1073/pnas.142288699
Davenport EC et al (2017) An essential role for the tetraspanin LHFPL4 in the cell-type-specific targeting and clustering of synaptic GABAA receptors. Cell Rep 21:70–83. https://doi.org/10.1016/j.celrep.2017.09.025
de Blas AL, Vitorica J, Friedrich P (1988) Localization of the GABAA receptor in the rat brain with a monoclonal antibody to the 57,000 Mr peptide of the GABAA receptor/benzodiazepine receptor/Cl- channel complex. J Neurosci 8:602–614. https://doi.org/10.1523/JNEUROSCI.08-02-00602.1988
Dixon C, Sah P, Lynch JW, Keramidas A (2014) GABAA receptor alpha and gamma subunits shape synaptic currents via different mechanisms. J Biol Chem 289:5399–5411. https://doi.org/10.1074/jbc.M113.514695
Essrich C, Lorez M, Benson JA, Fritschy JM, Luscher B (1998) Postsynaptic clustering of major GABAA receptor subtypes requires the gamma 2 subunit and gephyrin. Nat Neurosci 1:563–571. https://doi.org/10.1038/2798
Ewert M, de Blas AL, Mohler H, Seeburg PH (1992) A prominent epitope on GABAA receptors is recognized by two different monoclonal antibodies. Brain Res 569:57–62. https://doi.org/10.1016/0006-8993(92)90368-j
Farrar SJ, Whiting PJ, Bonnert TP, McKernan RM (1999) Stoichiometry of a ligand-gated ion channel determined by fluorescence energy transfer. J Biol Chem 274:10100–10104. https://doi.org/10.1074/jbc.274.15.10100
Fekete CD, Chiou TT, Miralles CP, Harris RS, Fiondella CG, Loturco JJ, De Blas AL (2015) In vivo clonal overexpression of neuroligin 3 and neuroligin 2 in neurons of the rat cerebral cortex: differential effects on GABAergic synapses and neuronal migration. J Comp Neurol 523:1359–1378. https://doi.org/10.1002/cne.23740
Fekete CD et al (2017) vivo transgenic expression of collybistin in neurons of the rat cerebral cortex. J Comp Neurol 525:1291–1311. https://doi.org/10.1002/cne.24137
Fruh S, Tyagarajan SK, Campbell B, Bosshard G, Fritschy JM (2018) The catalytic function of the gephyrin-binding protein IQSEC3 regulates neurotransmitter-specific matching of pre- and post-synaptic structures in primary hippocampal cultures. J Neurochem 147:477–494. https://doi.org/10.1111/jnc.14572
Fukaya M et al (2011) SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses. J Neurochem 116:1122–1137. https://doi.org/10.1111/j.1471-4159.2010.07167.x
Ge Y et al (2018) Clptm1 limits forward trafficking of GABAA receptors to scale inhibitory synaptic strength. Neuron 97:596-610 e598. https://doi.org/10.1016/j.neuron.2017.12.038
Gerrow K, Triller A (2014) GABAA receptor subunit composition and competition at synapses are tuned by GABAB receptor activity. Mol Cell Neurosci 60:97–107. https://doi.org/10.1016/j.mcn.2014.04.001
Ghosh H et al (2016) Several posttranslational modifications act in concert to regulate gephyrin scaffolding and GABAergic transmission. Nat Commun 7:13365. https://doi.org/10.1038/ncomms13365
Giesemann T et al (2003) Complex formation between the postsynaptic scaffolding protein gephyrin, profilin, and Mena: a possible link to the microfilament system. J Neurosci 23:8330–8339. https://doi.org/10.1523/JNEUROSCI.23-23-08330.2003
Groeneweg FL, Trattnig C, Kuhse J, Nawrotzki RA, Kirsch J (2018) Gephyrin: a key regulatory protein of inhibitory synapses and beyond histochem. Cell Biol 150:489–508. https://doi.org/10.1007/s00418-018-1725-2
Grosskreutz Y, Hermann A, Kins S, Fuhrmann JC, Betz H, Kneussel M (2001) Identification of a gephyrin-binding motif in the GDP/GTP exchange factor collybistin. Biol Chem 382:1455–1462. https://doi.org/10.1515/BC.2001.179
Grunewald N et al (2018) Sequences flanking the gephyrin-binding site of GlyRbeta tune receptor stabilization at synapses. eNeuro. https://doi.org/10.1523/ENEURO.0042-17.2018
Hannan S, Smart TG (2018) Cell surface expression of homomeric GABAA receptors depends on single residues in subunit transmembrane domains. J Biol Chem 293:13427–13439. https://doi.org/10.1074/jbc.RA118.002792
Harvey K et al (2004) The GDP-GTP exchange factor collybistin: an essential determinant of neuronal gephyrin clustering. J Neurosci 24:5816–5826. https://doi.org/10.1523/JNEUROSCI.1184-04.2004
Hausrat TJ et al (2015) Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory. Nat Commun 6:6872. https://doi.org/10.1038/ncomms7872
Heller EA et al (2012) The biochemical anatomy of cortical inhibitory synapses. PLoS ONE 7:e39572. https://doi.org/10.1371/journal.pone.0039572
Hines RM, Davies PA, Moss SJ, Maguire J (2012) Functional regulation of GABAA receptors in nervous system pathologies. Curr Opin Neurobiol 22:552–558. https://doi.org/10.1016/j.conb.2011.10.007
Hines RM et al (2018) Developmental seizures and mortality result from reducing GABAA receptor alpha2-subunit interaction with collybistin. Nat Commun 9:3130. https://doi.org/10.1038/s41467-018-05481-1
Jacob TC (2019) Neurobiology and therapeutic potential of alpha5-GABA type A receptors. Front Mol Neurosci 12:179. https://doi.org/10.3389/fnmol.2019.00179
Jacob TC, Moss SJ, Jurd R (2008) GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition. Nat Rev Neurosci 9:331–343. https://doi.org/10.1038/nrn2370
Jechlinger M, Pelz R, Tretter V, Klausberger T, Sieghart W (1998) Subunit composition and quantitative importance of hetero-oligomeric receptors: GABAA receptors containing alpha6 subunits. J Neurosci 18:2449–2457. https://doi.org/10.1523/JNEUROSCI.18-07-02449.1998
Jin H, Chiou TT, Serwanski DR, Miralles CP, Pinal N, De Blas AL (2014) Ring finger protein 34 (RNF34) interacts with and promotes gamma-aminobutyric acid type-A receptor degradation via ubiquitination of the gamma2 subunit. J Biol Chem 289:29420–29436. https://doi.org/10.1074/jbc.M114.603068
Kalscheuer VM et al (2009) A balanced chromosomal translocation disrupting ARHGEF9 is associated with epilepsy, anxiety, aggression, and mental retardation. Hum Mutat 30:61–68. https://doi.org/10.1002/humu.20814
Kasaragod VB, Schindelin H (2018) Structure-function relationships of glycine and GABAA receptors and their interplay with the scaffolding protein gephyrin. Front Mol Neurosci 11:317. https://doi.org/10.3389/fnmol.2018.00317
Kasaragod VB, Schindelin H (2019) Structure of heteropentameric GABAA receptors and receptor-anchoring properties of gephyrin. Front Mol Neurosci 12:191. https://doi.org/10.3389/fnmol.2019.00191
Kasugai Y et al (2010) Quantitative localisation of synaptic and extrasynaptic GABAA receptor subunits on hippocampal pyramidal cells by freeze-fracture replica immunolabelling. Eur J Neurosci 32:1868–1888. https://doi.org/10.1111/j.1460-9568.2010.07473.x
Khan ZU, Gutierrez A, Mehta AK, Miralles CP, De Blas AL (1996) The alpha 4 subunit of the GABAA receptors from rat brain and retina. Neuropharmacology 35:1315–1322. https://doi.org/10.1016/s0028-3908(96)00033-0
Kins S, Betz H, Kirsch J (2000) Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin. Nat Neurosci 3:22–29. https://doi.org/10.1038/71096
Kirsch J, Kuhse J, Betz H (1995) Targeting of glycine receptor subunits to gephyrin-rich domains in transfected human embryonic kidney cells. Mol Cell Neurosci 6:450–461. https://doi.org/10.1006/mcne.1995.1033
Kirsch J, Langosch D, Prior P, Littauer UZ, Schmitt B, Betz H (1991) The 93-kDa glycine receptor-associated protein binds to tubulin. J Biol Chem 266:22242–22245
Kittler JT, Delmas P, Jovanovic JN, Brown DA, Smart TG, Moss SJ (2000) Constitutive endocytosis of GABAA receptors by an association with the adaptin AP2 complex modulates inhibitory synaptic currents in hippocampal neurons. J Neurosci 20:7972–7977. https://doi.org/10.1523/JNEUROSCI.20-21-07972.2000
Kneussel M, Brandstatter JH, Laube B, Stahl S, Muller U, Betz H (1999) Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient mice. J Neurosci 19:9289–9297. https://doi.org/10.1523/JNEUROSCI.19-21-09289.1999
Kneussel M, Brandstatter JH, Gasnier B, Feng G, Sanes JR, Betz H (2001) Gephyrin-independent clustering of postsynaptic GABA(A) receptor subtypes. Mol Cell Neurosci 17:973–982. https://doi.org/10.1006/mcne.2001.0983
Kowalczyk S, Winkelmann A, Smolinsky B, Forstera B, Neundorf I, Schwarz G, Meier JC (2013) Direct binding of GABAA receptor beta2 and beta3 subunits to gephyrin. Eur J Neurosci 37:544–554. https://doi.org/10.1111/ejn.12078
Krueger-Burg D, Papadopoulos T, Brose N (2017) Organizers of inhibitory synapses come of age. Curr Opin Neurobiol 45:66–77. https://doi.org/10.1016/j.conb.2017.04.003
Lagrange AH, Hu N, Macdonald RL (2018) GABA beyond the synapse: defining the subtype-specific pharmacodynamics of non-synaptic GABAA receptors. J Physiol 596:4475–4495. https://doi.org/10.1113/JP276187
Laverty D et al (2019) Cryo-EM structure of the human alpha1beta3gamma2 GABAA receptor in a lipid bilayer. Nature 565:516–520. https://doi.org/10.1038/s41586-018-0833-4
Levi S, Logan SM, Tovar KR, Craig AM (2004) Gephyrin is critical for glycine receptor clustering but not for the formation of functional GABAergic synapses in hippocampal neurons. J Neurosci 24:207–217. https://doi.org/10.1523/JNEUROSCI.1661-03.2004
Li RW, Yu W, Christie S, Miralles CP, Bai J, Loturco JJ, De Blas AL (2005) Disruption of postsynaptic GABA receptor clusters leads to decreased GABAergic innervation of pyramidal neurons. J Neurochem 95:756–770. https://doi.org/10.1111/j.1471-4159.2005.03426.x
Li X, Serwanski DR, Miralles CP, Bahr BA, De Blas AL (2007) Two pools of Triton X-100-insoluble GABA(A) receptors are present in the brain, one associated to lipid rafts and another one to the post-synaptic GABAergic complex. J Neurochem 102:1329–1345. https://doi.org/10.1111/j.1471-4159.2007.04635.x
Li X, Serwanski DR, Miralles CP, Nagata K, De Blas AL (2009) Septin 11 is present in GABAergic synapses and plays a functional role in the cytoarchitecture of neurons and GABAergic synaptic connectivity. J Biol Chem 284:17253–17265. https://doi.org/10.1074/jbc.M109.008870
Li Y, Serwanski DR, Miralles CP, Fiondella CG, Loturco JJ, Rubio ME, De Blas AL (2010) Synaptic and nonsynaptic localization of protocadherin-gammaC5 in the rat brain. J Comp Neurol 518:3439–3463. https://doi.org/10.1002/cne.22390
Li Y et al (2012) Molecular and functional interaction between protocadherin-gammaC5 and GABAA receptors. J Neurosci 32:11780–11797. https://doi.org/10.1523/JNEUROSCI.0969-12.2012
Loebrich S, Bahring R, Katsuno T, Tsukita S, Kneussel M (2006) Activated radixin is essential for GABAA receptor alpha5 subunit anchoring at the actin cytoskeleton. EMBO J 25:987–999. https://doi.org/10.1038/sj.emboj.7600995
Loh KH et al (2016) Proteomic analysis of unbounded cellular compartments: synaptic clefts. Cell 166(1295–1307):e1221. https://doi.org/10.1016/j.cell.2016.07.041
Ludolphs M et al (2016) Specificity of collybistin-phosphoinositide interactions: impact of the individual protein domains. J Biol Chem 291:244–254. https://doi.org/10.1074/jbc.M115.673400
Luscher B, Fuchs T, Kilpatrick CL (2011) GABAA receptor trafficking-mediated plasticity of inhibitory synapses. Neuron 70:385–409. https://doi.org/10.1016/j.neuron.2011.03.024
Magnin E, Francavilla R, Amalyan S, Gervais E, David LS, Luo X, Topolnik L (2019) Input-specific synaptic location and function of the alpha5 GABAA receptor subunit in the mouse CA1 hippocampal neurons. J Neurosci 39:788–801. https://doi.org/10.1523/JNEUROSCI.0567-18.2018
Maric HM, Mukherjee J, Tretter V, Moss SJ, Schindelin H (2011) Gephyrin-mediated gamma-aminobutyric acid type A and glycine receptor clustering relies on a common binding site. J Biol Chem 286:42105–42114. https://doi.org/10.1074/jbc.M111.303412
Maric HM, Kasaragod VB, Schindelin H (2014) Modulation of gephyrin-glycine receptor affinity by multivalency. ACS Chem Biol 9:2554–2562. https://doi.org/10.1021/cb500303a
Maric HM, Kasaragod VB, Haugaard-Kedstrom L, Hausrat TJ, Kneussel M, Schindelin H, Stromgaard K (2015) Design and synthesis of high-affinity dimeric inhibitors targeting the interactions between gephyrin and inhibitory neurotransmitter receptors. Angew Chem Int Ed Engl 54:490–494. https://doi.org/10.1002/anie.201409043
Martenson JS, Yamasaki T, Chaudhury NH, Albrecht D, Tomita S (2017) Assembly rules for GABAA receptor complexes in the brain. Elife. https://doi.org/10.7554/eLife.27443
Masiulis S et al (2019) GABAA receptor signalling mechanisms revealed by structural pharmacology. Nature 565:454–459. https://doi.org/10.1038/s41586-018-0832-5
Meyer G, Kirsch J, Betz H, Langosch D (1995) Identification of a gephyrin binding motif on the glycine receptor beta subunit. Neuron 15:563–572. https://doi.org/10.1016/0896-6273(95)90145-0
Miralles CP, Li M, Mehta AK, Khan ZU, De Blas AL (1999) Immunocytochemical localization of the beta(3) subunit of the gamma-aminobutyric acid(A) receptor in the rat brain. J Comp Neurol 413:535–548. https://doi.org/10.1002/(SICI)1096-9861(19991101)413:4%3c535::AID-CNE4%3e3.0.CO;2-T
Mortensen M, Smart TG (2006) Extrasynaptic alphabeta subunit GABAA receptors on rat hippocampal pyramidal neurons. J Physiol 577:841–856. https://doi.org/10.1113/jphysiol.2006.117952
Mortensen M, Patel B, Smart TG (2011) GABA Potency at GABA(A) receptors found in synaptic and extrasynaptic zones. Front Cell Neurosci 6:1. https://doi.org/10.3389/fncel.2012.00001
Muir J, Arancibia-Carcamo IL, MacAskill AF, Smith KR, Griffin LD, Kittler JT (2010) NMDA receptors regulate GABAA receptor lateral mobility and clustering at inhibitory synapses through serine 327 on the gamma2 subunit. Proc Natl Acad Sci USA 107:16679–16684. https://doi.org/10.1073/pnas.1000589107
Mukherjee J et al (2011) The residence time of GABA(A)Rs at inhibitory synapses is determined by direct binding of the receptor alpha1 subunit to gephyrin. J Neurosci 31:14677–14687. https://doi.org/10.1523/JNEUROSCI.2001-11.2011
Nakamura Y et al (2016) Proteomic characterization of inhibitory synapses using a novel pHluorin-tagged gamma-aminobutyric acid receptor, type A (GABAA), alpha2 subunit knock-in mouse. J Biol Chem 291:12394–12407. https://doi.org/10.1074/jbc.M116.724443
Nathanson AJ et al (2019) Identification of a core amino acid motif within the alpha subunit of GABAARs that promotes inhibitory synaptogenesis and resilience to seizures. Cell Rep 28(670–681):e678. https://doi.org/10.1016/j.celrep.2019.06.014
Nguyen QA, Nicoll RA (2018) The GABAA receptor beta subunit is required for inhibitory transmission. Neuron 98(718–725):e713. https://doi.org/10.1016/j.neuron.2018.03.046
Nusser Z, Sieghart W, Somogyi P (1998) Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells. J Neurosci 18:1693–1703. https://doi.org/10.1523/JNEUROSCI.18-05-01693.1998
Olsen RW, Sieghart W (2008) International Union of Pharmacology. LXX. Subtypes of gamma-aminobutyric acid(A) receptors: classification on the basis of subunit composition, pharmacology, and function. Update Pharmacol Rev 60:243–260. https://doi.org/10.1124/pr.108.00505
Panzanelli P et al (2011) Distinct mechanisms regulate GABAA receptor and gephyrin clustering at perisomatic and axo-axonic synapses on CA1 pyramidal cells. J Physiol 589:4959–4980. https://doi.org/10.1113/jphysiol.2011.216028
Papadopoulos T et al (2007) Impaired GABAergic transmission and altered hippocampal synaptic plasticity in collybistin-deficient mice. EMBO J 26:3888–3899. https://doi.org/10.1038/sj.emboj.7601819
Papadopoulos T, Eulenburg V, Reddy-Alla S, Mansuy IM, Li Y, Betz H (2008) Collybistin is required for both the formation and maintenance of GABAergic postsynapses in the hippocampus. Mol Cell Neurosci 39:161–169. https://doi.org/10.1016/j.mcn.2008.06.006
Papadopoulos T et al (2017) Endosomal phosphatidylinositol 3-phosphate promotes gephyrin clustering and GABAergic neurotransmission at inhibitory postsynapses. J Biol Chem 292:1160–1177. https://doi.org/10.1074/jbc.M116.771592
Patrizi A, Viltono L, Frola E, Harvey K, Harvey RJ, Sassoe-Pognetto M (2012) Selective localization of collybistin at a subset of inhibitory synapses in brain circuits. J Comp Neurol 520:130–141. https://doi.org/10.1002/cne.22702
Petrini EM, Barberis A (2014) Diffusion dynamics of synaptic molecules during inhibitory postsynaptic plasticity. Front Cell Neurosci 8:300. https://doi.org/10.3389/fncel.2014.00300
Pirker S, Schwarzer C, Wieselthaler A, Sieghart W, Sperk G (2000) GABA(A) receptors: immunocytochemical distribution of 13 subunits in the adult rat brain. Neuroscience 101:815–850. https://doi.org/10.1016/s0306-4522(00)00442-5
Pizzarelli R, Griguoli M, Zacchi P, Petrini EM, Barberis A, Cattaneo A, Cherubini E (2019) Tuning GABAergic inhibition: Gephyrin molecular organization and functions. Neuroscience. https://doi.org/10.1016/j.neuroscience.2019.07.036
Poulopoulos A et al (2009) Neuroligin 2 drives postsynaptic assembly at perisomatic inhibitory synapses through gephyrin and collybistin. Neuron 63:628–642. https://doi.org/10.1016/j.neuron.2009.08.023
Prior P et al (1992) Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. Neuron 8:1161–1170. https://doi.org/10.1016/0896-6273(92)90136-2
Reid T, Bathoorn A, Ahmadian MR, Collard JG (1999) Identification and characterization of hPEM-2, a guanine nucleotide exchange factor specific for Cdc42. J Biol Chem 274:33587–33593. https://doi.org/10.1074/jbc.274.47.33587
Riquelme R, Miralles CP, De Blas AL (2002) Bergmann glia GABA(A) receptors concentrate on the glial processes that wrap inhibitory synapses. J Neurosci 22:10720–10730. https://doi.org/10.1523/JNEUROSCI.22-24-10720.2002
Saiepour L, Fuchs C, Patrizi A, Sassoe-Pognetto M, Harvey RJ, Harvey K (2010) Complex role of collybistin and gephyrin in GABAA receptor clustering. J Biol Chem 285:29623–29631. https://doi.org/10.1074/jbc.M110.121368
Sakagami H, Katsumata O, Hara Y, Tamaki H, Watanabe M, Harvey RJ, Fukaya M (2013) Distinct synaptic localization patterns of brefeldin A-resistant guanine nucleotide exchange factors BRAG2 and BRAG3 in the mouse retina. J Comp Neurol 521:860–876. https://doi.org/10.1002/cne.23206
Schulz JM, Knoflach F, Hernandez MC, Bischofberger J (2018) Dendrite-targeting interneurons control synaptic NMDA-receptor activation via nonlinear alpha5-GABAA receptors. Nat Commun 9:3576. https://doi.org/10.1038/s41467-018-06004-8
Schweizer C, Balsiger S, Bluethmann H, Mansuy IM, Fritschy JM, Mohler H, Luscher B (2003) The gamma 2 subunit of GABA(A) receptors is required for maintenance of receptors at mature synapses. Mol Cell Neurosci 24:442–450. https://doi.org/10.1016/S1044-7431(03)00202-1
Serwanski DR, Miralles CP, Christie SB, Mehta AK, Li X, De Blas AL (2006) Synaptic and nonsynaptic localization of GABAA receptors containing the alpha5 subunit in the rat brain. J Comp Neurol 499:458–470. https://doi.org/10.1002/cne.21115
Sieghart W, Sperk G (2002) Subunit composition, distribution and function of GABA(A) receptor subtypes. Curr Top Med Chem 2:795–816. https://doi.org/10.2174/1568026023393507
Sinkkonen ST, Vekovischeva OY, Moykkynen T, Ogris W, Sieghart W, Wisden W, Korpi ER (2004) Behavioural correlates of an altered balance between synaptic and extrasynaptic GABAAergic inhibition in a mouse model. Eur J Neurosci 20:2168–2178. https://doi.org/10.1111/j.1460-9568.2004.03684.x
Specht CG (2019) Fractional occupancy of synaptic binding sites and the molecular plasticity of inhibitory synapses. Neuropharmacology. https://doi.org/10.1016/j.neuropharm.2019.01.008
Sun C, Sieghart W, Kapur J (2004) Distribution of alpha1, alpha4, gamma2, and delta subunits of GABAA receptors in hippocampal granule cells. Brain Res 1029:207–216. https://doi.org/10.1016/j.brainres.2004.09.056
Sun MY et al (2018) Chemogenetic isolation reveals synaptic contribution of delta GABAA Receptors In Mouse Dentate Granule Neurons. J Neurosci 38:8128–8145. https://doi.org/10.1523/JNEUROSCI.0799-18.2018
Tretter V, Ehya N, Fuchs K, Sieghart W (1997) Stoichiometry and assembly of a recombinant GABAA receptor subtype. J Neurosci 17:2728–2737. https://doi.org/10.1523/JNEUROSCI.17-08-02728.1997
Tretter V, Jacob TC, Mukherjee J, Fritschy JM, Pangalos MN, Moss SJ (2008) The clustering of GABA(A) receptor subtypes at inhibitory synapses is facilitated via the direct binding of receptor alpha 2 subunits to gephyrin. J Neurosci 28:1356–1365. https://doi.org/10.1523/JNEUROSCI.5050-07.2008
Tretter V et al (2011) Molecular basis of the gamma-aminobutyric acid A receptor alpha3 subunit interaction with the clustering protein gephyrin. J Biol Chem 286:37702–37711. https://doi.org/10.1074/jbc.M111.291336
Tretter V, Mukherjee J, Maric HM, Schindelin H, Sieghart W, Moss SJ (2012) Gephyrin, the enigmatic organizer at GABAergic synapses. Front Cell Neurosci 6:23. https://doi.org/10.3389/fncel.2012.00023
Tyagarajan SK, Fritschy JM (2014) Gephyrin: a master regulator of neuronal function? Nat Rev Neurosci 15:141–156. https://doi.org/10.1038/nrn3670
Tyagarajan SK, Ghosh H, Harvey K, Fritschy JM (2011) Collybistin splice variants differentially interact with gephyrin and Cdc42 to regulate gephyrin clustering at GABAergic synapses. J Cell Sci 124:2786–2796. https://doi.org/10.1242/jcs.086199
Uezu A et al (2016) Identification of an elaborate complex mediating postsynaptic inhibition. Science 353:1123–1129. https://doi.org/10.1126/science.aag0821
Um JW et al (2016) IQ motif and SEC7 domain-containing protein 3 (IQSEC3) interacts with gephyrin to promote inhibitory synapse formation. J Biol Chem 291:10119–10130. https://doi.org/10.1074/jbc.M115.712893
Varoqueaux F, Jamain S, Brose N (2004) Neuroligin 2 is exclusively localized to inhibitory synapses. Eur J Cell Biol 83:449–456. https://doi.org/10.1078/0171-9335-00410
Vitorica J, Park D, Chin G, de Blas AL (1988) Monoclonal antibodies and conventional antisera to the GABAA receptor/benzodiazepine receptor/Cl- channel complex. J Neurosci 8:615–622. https://doi.org/10.1523/JNEUROSCI.08-02-00615.1988
Wei W, Zhang N, Peng Z, Houser CR, Mody I (2003) Perisynaptic localization of delta subunit-containing GABA(A) receptors and their activation by GABA spillover in the mouse dentate gyrus. J Neurosci 23:10650–10661. https://doi.org/10.1523/JNEUROSCI.23-33-10650.2003
Wu X et al (2012) gamma-Aminobutyric acid type A (GABAA) receptor alpha subunits play a direct role in synaptic versus extrasynaptic targeting. J Biol Chem 287:27417–27430. https://doi.org/10.1074/jbc.M112.360461
Wu M, Tian HL, Liu X, Lai JHC, Du S, Xia J (2018) Impairment of inhibitory synapse formation and motor behavior in mice lacking the NL2 binding partner LHFPL4/GARLH4. Cell Rep 23:1691–1705. https://doi.org/10.1016/j.celrep.2018.04.015
Xiang S et al (2006) The crystal structure of Cdc42 in complex with collybistin II, a gephyrin-interacting guanine nucleotide exchange factor. J Mol Biol 359:35–46. https://doi.org/10.1016/j.jmb.2006.03.019
Yamasaki T, Hoyos-Ramirez E, Martenson JS, Morimoto-Tomita M, Tomita S (2017) GARLH family proteins stabilize GABAA receptors at synapses. Neuron 93(1138–1152):e1136. https://doi.org/10.1016/j.neuron.2017.02.023
Yu W, De Blas AL (2008) Gephyrin expression and clustering affects the size of glutamatergic synaptic contacts. J Neurochem 104:830–845. https://doi.org/10.1111/j.1471-4159.2007.05014.x
Yu W, Jiang M, Miralles CP, Li RW, Chen G, de Blas AL (2007) Gephyrin clustering is required for the stability of GABAergic synapses. Mol Cell Neurosci 36:484–500. https://doi.org/10.1016/j.mcn.2007.08.008
Yu W, Charych EI, Serwanski DR, Li RW, Ali R, Bahr BA, De Blas AL (2008) Gephyrin interacts with the glutamate receptor interacting protein 1 isoforms at GABAergic synapses. J Neurochem 105:2300–2314. https://doi.org/10.1111/j.1471-4159.2008.05311.x
Acknowledgements
We thank Profs. K. Harvey (UCL School of Pharmacy, London, UK) and R.J. Harvey (University of the Sunshine Coast, Sippy Downs, Australia) for providing the CB plasmids and Profs. J.M. Fritschy and S.K. Tyagarajan (Institute of Pharmacology and Toxicology, University of Zurich, Switzerland) for the EGFP-Geph plasmid. We also thank the late Dr Peter Seeburg (Max Planck-Institute for Medical Research, Heidelberg) for GluA1, GluA2, GluN1, GluN2B and mycGluN2B plasmids. We also thank Ms. Toni Vella for her participation in some experiments.
Funding
This research was supported by the NIH-NINDS Grant R01NS038752 to A.L.D. and a University of Connecticut Research Incentive Program grant to A.L.D.
Author information
Authors and Affiliations
Contributions
ALD involved in the study concept and design. SG, T-TC and KK participated in acquisition of data. SG, T-TC, KK and ALD involved in analysis and interpretation of data. ALD drafted the manuscript. SG and ALD involved in critical revision of the manuscript for important intellectual content. SG participated in statistical analysis. SG and ALD supervised the study. ALD obtained the funding.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no conflicts of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The original online version of this article was revised: the figures in supplementary material were swapped and now it has been corrected.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary file1 (TIF 3686 KB)
Fig. S1. (A): HEK293 cells were co-transfected with EGFP-Geph and HA-CB2SH3-; Fixed and permeabilized; Ms anti-HA (red). (B-C) HEK293 cells were co-transfected with EGFP-Geph and myc-CB2SH3- and either fixed and non-permeabilized (no Triton X-100; B), or fixed and permeabilized (C); Ms anti-Geph (red), Rb anti-CB (blue). Scale bar = 10 μm for A and 12 μm for B&C.
Supplementary file2 (TIF 5045 KB)
Fig. S2. (A-B): Triple-label immunofluorescence of cells co-transfected with α1, β3, γ2 and either EGFP-Geph (A) or myc-CB2SH3- (B). Live-cell two-step capping in A with no permeabilization. Live cell two-step capping with anti-GABAAR subunit antibodies followed by fixation and permeabilization for anti-myc labeling. Rb anti- γ2 (red), and either EGFP-Geph fluorescence (A; green) or Ms anti-myc (B; blue). (C) Triple-label immunofluorescence of cells co-transfected with α1, β3, and γ2. Fixed and permeabilized; GP anti-α1 (green); Ms anti-β2/3(blue); Rb anti-γ2 (red). (D-E): HEK293 cells co-transfected with α1, β3, γ2, EGFP-Geph and myc-CB2SH3-. Fixed and permeabilized; EGFP fluorescence (green); Ms anti-myc (blue in D); Ms anti-β2/3 (blue in E); Rb anti-γ2 (red). Scale bar = 11.2 μm for A,B & E; 25 μm for C; 13.4 μm for D.
Supplementary file3 (TIF 5618 KB)
Fig. S3. (A): Triple-label fluorescence of fixed and permeabilized cells that were co-transfected with α1, β3, EGFP-Geph and myc-CB2SH3-. Rb anti-α1 (red); EGFP fluorescence (green); Ms anti-myc (blue). (B): Triple-label fluorescence of cells that were co-transfected with α1, β3, EGFP-Geph and myc-CB2SH3-. Surface labeling was done by live-cell incubation with Abs. Ms anti-β2/3 (red); Rb anti-α1 (blue); EGFP-Geph fluorescence (green). (C) Triple-label fluorescence of fixed and permeabilized cells that were co-transfected with β3, EGFP-Geph and myc-CB2SH3-. Ms anti-β2/3; EGFP fluorescence (green); Rb anti-myc (blue). (D) Triple-label fluorescence of cells that were co-transfected with β3, EGFP-Geph and myc-CB2SH3-. Surface labeling was done by live-cell incubation with Abs. Ms anti-β2/3 (blue); EGFP fluorescence (green); Rb anti-CB (red). (E): Double-label fluorescence of fixed and permeabilized cells that were co-transfected with γ2, EGFP-Geph and myc-CB2SH3-. EGFP-Geph fluorescence (green); Rb anti-γ2 (red). (F): Double-label fluorescence of fixed and permeabilized cells co-transfected with α1, EGFP-Geph and myc-CB2SH3-. Rb anti-α1 (red); EGFP-Geph fluorescence (green). Scale bar = 23.4 µm for A; 14.4 µm for B; 18 µm for C&D to P; 10 µm for E&F
Supplementary file4 (TIF 5839 KB)
Fig. S4. (A-B): Cells transfected with α4 (A) or mycδ (B) react with Rb anti-α4 or Ms anti-myc, respectively, in fixed and permeabilized cells. (C): Cells were co-transfected with α4, β3-EGFP and mycδ. Live cell incubation with Rb anti-α4 (blue), Ms anti-myc (red) and β3-EGFP fluorescence (green) shows capping and co-localization of the three of GABAAR subunits at the cell surface. Second and fourth panels show overlays of blue and red, and green and red, respectively. (D&E): Cells were co-transfected with α4, β3, and mycδ, EGFP-Geph and HA-CB2SH3-. Cells were fixed and permeabilized before incubation with Ms anti-myc (red) and Rb α4 (blue). Most cells show little to no co-localization (D) while few show some co-localization (E). (F): Cells were co-transfected with α4 and β3. Fixed and non-permeabilized transfected cells were incubated with Rb anti-α4 (red); Ms anti-β2/3 (blue). (G&H): HEK293 cells were co-transfected with α5, β3-EGFP, mycγ2. (G): Cells were fixed and permeabilized before incubation with antibodies. (H): Live cell incubation of transfected cells with Rb anti-α5 (red) and Ms anti-myc (blue) shows capping of the GABAARs including β3-EGFP (green) at the cell surface. Scale bar = 10 µm for A&B; 14 µm for C; 11.3 µm for D; 5.6 µm for E; 7.5 µm for F; 17.2 µm for G; and 6.8 µm for H
Supplementary file5 (TIF 2370 KB)
Fig. S5. Surface AMPA and NMDA glutamate receptors do not associate with submembranous Geph/CB2SH3- clusters. Triple-label fluorescence. (A-C): Cells were co-transfected with GluA1, GluA2, EGFP-Geph and HA-CB2SH3-. In A cells were fixed and permeabilized before labeling. Ms anti-GluA2 (red) and Rb anti-GluA1 (blue); In B-C, immunolabelling was done after fixation without permeabilization. (B) surface labeling of GluA2 (red) and GluA1 (blue). There is no surface labeling of GluA1 because Rb anti-GluA1 recognizes an intracellular epitope. (C) Confocal image overlay of GluA2 (blue) and EGFP-Geph (green). (D-F): Cells were co-transfected with GluN1, mycGluN2B, EGFP-Geph and HA-CB2SH3-. In D, cells were fixed and permeabilized before labeling. In E-F immunolabelling was done after fixation without permeabilization. Rb anti-myc (red) and EGFP-Geph fluorescence (green); J shows a confocal image. Scale bar = 6.6 µm for A; 10 µm for B&D and 8.3 µm for C,E-F
Rights and permissions
About this article
Cite this article
George, S., Chiou, TT., Kanamalla, K. et al. Recruitment of Plasma Membrane GABA-A Receptors by Submembranous Gephyrin/Collybistin Clusters. Cell Mol Neurobiol 42, 1585–1604 (2022). https://doi.org/10.1007/s10571-021-01050-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10571-021-01050-1