Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit Counting

The subunit stoichiometry of heteromeric glycine-gated channels determines fundamental properties of these key inhibitory neurotransmitter receptors; however, the ratio of α1- to β-subunits per receptor remains controversial. We used single-molecule imaging and stepwise photobleaching in Xenopus oocytes to directly determine the subunit stoichiometry of a glycine receptor to be 3α1:2β. This approach allowed us to determine the receptor stoichiometry in mixed populations consisting of both heteromeric and homomeric channels, additionally revealing the quantitative proportions for the two populations.

[1]  R. Werman,et al.  The distribution of glycine in cat spinal cord and roots. , 1965, Life sciences.

[2]  A technical consideration concerning the removal of oocyte vitelline membranes for patch clamp recording. , 2004, Biochemical and biophysical research communications.

[3]  J. Lynch,et al.  Native glycine receptor subtypes and their physiological roles , 2009, Neuropharmacology.

[4]  M. Shapiro,et al.  Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[5]  N. Hussy,et al.  Structural difference between heteromeric somatic and homomeric axonal glycine receptors in the hypothalamo-neurohypophysial system , 2005, Neuroscience.

[6]  A. Triller,et al.  Gephyrin antisense oligonucleotides prevent glycine receptor clustering in spinal neurons , 1993, Nature.

[7]  Dieter Langosch,et al.  Identification of a gephyrin binding motif on the glycine receptor β subunit , 1995, Neuron.

[8]  E. Sigel,et al.  Subunit Arrangement of γ-Aminobutyric Acid Type A Receptors* , 2001, The Journal of Biological Chemistry.

[9]  Heinrich Betz,et al.  The β Subunit Determines the Ligand Binding Properties of Synaptic Glycine Receptors , 2005, Neuron.

[10]  E. Gundelfinger,et al.  The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors , 1987, Nature.

[11]  S. Dieudonné.  Glycinergic synaptic currents in Golgi cells of the rat cerebellum. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[12]  R. A. Davidoff,et al.  Inhibition of motoneurones by iontophoresis of glycine. , 1967, Nature.

[13]  Marco Beato,et al.  Single-Channel Behavior of Heteromeric α1β Glycine Receptors: An Attempt to Detect a Conformational Change before the Channel Opens , 2004, The Journal of Neuroscience.

[14]  J. Lynch,et al.  Molecular structure and function of the glycine receptor chloride channel. , 2004, Physiological reviews.

[15]  E. Speckmann,et al.  A concentration-clamp system allowing two-electrode voltage-clamp investigations in oocytes of Xenopus laevis , 1991, Journal of Neuroscience Methods.

[16]  Erik A. Rodriguez,et al.  Single-molecule imaging of a fluorescent unnatural amino acid incorporated into nicotinic receptors. , 2009, Biophysical journal.

[17]  T. Klausberger,et al.  Subunit Composition and Quantitative Importance of Hetero-oligomeric Receptors: GABAA Receptors Containing α6 Subunits , 1998, The Journal of Neuroscience.

[18]  H. Wässle,et al.  GlyR α3: An Essential Target for Spinal PGE2-Mediated Inflammatory Pain Sensitization , 2004, Science.

[19]  T. Mohandas,et al.  Alpha subunit variants of the human glycine receptor: primary structures, functional expression and chromosomal localization of the corresponding genes. , 1990, The EMBO journal.

[20]  L. Trussell,et al.  Reciprocal developmental regulation of presynaptic ionotropic receptors , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. L. Goldin.  Maintenance of Xenopus laevis and oocyte injection. , 1992, Methods in enzymology.

[22]  M. Ballivet,et al.  Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor , 1991, Nature.

[23]  D. Langosch,et al.  Conserved quaternary structure of ligand-gated ion channels: the postsynaptic glycine receptor is a pentamer. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[24]  G. Meyer,et al.  Identification of a gephyrin binding motif on the glycine receptor beta subunit. , 1995, Neuron.

[25]  L. Trussell,et al.  Presynaptic glycine receptors enhance transmitter release at a mammalian central synapse , 2001, Nature.

[26]  J. Bormann,et al.  The atypical M2 segment of the beta subunit confers picrotoxinin resistance to inhibitory glycine receptor channels. , 1992, The EMBO journal.

[27]  M. Tijssen,et al.  Startle syndromes , 2009 .

[28]  I. Putrenko,et al.  A Family of Acetylcholine-gated Chloride Channel Subunits in Caenorhabditis elegans* , 2005, Journal of Biological Chemistry.

[29]  Paul J. Groot-Kormelink,et al.  Stoichiometry of recombinant heteromeric glycine receptors revealed by a pore-lining region point mutation. , 2003, Receptors & channels.

[30]  P. Selvin,et al.  Counting bungarotoxin binding sites of nicotinic acetylcholine receptors in mammalian cells with high signal/noise ratios. , 2010, Biophysical journal.

[31]  J. Lynch,et al.  Molecular pharmacology of the glycine receptor chloride channel. , 2007, Current pharmaceutical design.

[32]  E. Isacoff,et al.  Subunit counting in membrane-bound proteins , 2007, Nature Methods.

[33]  Werner Sieghart,et al.  Stoichiometry and Assembly of a Recombinant GABAA Receptor Subtype , 1997, The Journal of Neuroscience.

[34]  Spencer S. Ericksen,et al.  Tandem couture: Cys-loop receptor concatamer insights and caveats. , 2007, Molecular neurobiology.

[35]  E. Sigel,et al.  Subunit Stoichiometry of Oligomeric Membrane Proteins: GABAA Receptors Isolated by Selective Immunoprecipitation from the Cell Surface , 1996, Neuropharmacology.

[36]  E. Sigel,et al.  Techniques: Use of concatenated subunits for the study of ligand-gated ion channels. , 2004, Trends in pharmacological sciences.

[37]  H. Betz,et al.  Assembly of the inhibitory glycine receptor: Identification of amino acid sequence motifs governing subunit stoichiometry , 1993, Neuron.