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An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors

Nature Structural & Molecular Biology volume 17, pages 90–98 (2010)Cite this article

Abstract

Cys-loop receptors are pentameric ligand-gated ion channels (pLGICs) that mediate fast synaptic transmission. Here functional pentameric assembly of truncated fragments comprising the ligand-binding N-terminal ectodomains and the first three transmembrane helices, M1–M3, of both the inhibitory glycine receptor (GlyR) α1 and the 5HT3A receptor subunits was found to be rescued by coexpressing the complementary fourth transmembrane helix, M4. Alanine scanning identified multiple aromatic residues in M1, M3 and M4 as key determinants of GlyR assembly. Homology modeling and molecular dynamics simulations revealed that these residues define an interhelical aromatic network, which we propose determines the geometry of M1–M4 tetrahelical packing such that nascent pLGIC subunits must adopt a closed fivefold symmetry. Because pLGIC ectodomains form random nonstoichiometric oligomers, proper pentameric assembly apparently depends on intersubunit interactions between extracellular domains and intrasubunit interactions between transmembrane segments.

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Figure 1: Oligomeric structures of α1 GlyR truncation mutants.
Figure 2: Reconstitution of pentamer formation upon coexpression of N- and C-terminally truncated GlyR α1 subunit fragments.
Figure 3: Reconstitution of α1 GlyR and 5HT3A receptor function from complementary homotypic fragments.
Figure 4: Effect of single alanine substitutions in M4 on assembly, surface expression and function of the α1 GlyR.
Figure 5: Location of membrane-embedded aromatic residues crucial for homopentameric assembly of the α1 GlyR.

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Acknowledgements

We thank U. Braam for expert help in generating cDNA constructs, G. Drwal for electrophysiological recording and F. Pult for sharing with us the data of her M.D. work on C-terminal N-glycosylation. Supported by Deutsche Forschungsgemeinschaft (La1086/5-3; Schm536/4-3), Fonds der Chemischen Industrie (H.B.), the Hertie Foundation (B.L.), the Alexander von Humboldt Foundation (H.B., V.T.), Deutscher Akademischer Austauschdienst (D.K.) and the Russian Academy of Sciences grant “Molecular and Cell Biology” (V.T., D.K.).

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Authors and Affiliations

  1. Molecular Pharmacology, RWTH Aachen University of Aachen, Aachen, Germany

    Svenja Haeger, Silvia Detro-Dassen, Niklas Lang & Günther Schmalzing

  2. Department of Neurochemistry, Max Planck Institute for Brain Research, Frankfurt am Main, Germany

    Dmitry Kuzmin, Heinrich Betz & Bodo Laube

  3. Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia

    Dmitry Kuzmin & Victor Tsetlin

  4. Department Molekulare und zelluläre Neurophysiologie, Technische Universität Darmstadt, Darmstadt, Germany

    Michael Kilb & Bodo Laube

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  1. Svenja Haeger
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Contributions

S.H. designed and generated DNA constructs and performed biochemical experiments; D.K. performed homology modeling and molecular dynamics simulations and analyzed the data; S.D.-D. synthesized cRNAs, generated DNA constructs and performed biochemical experiments; N.L. generated mutants and performed biochemical experiments; M.K. performed electrophysiological recordings; V.T. supervised homology modeling and analyzed data; H.B. designed experiments and wrote the paper; B.L. designed experiments, performed electrophysiological recordings, analyzed data and wrote the paper; G.S. designed constructs and biochemical experiments, analyzed the data and wrote the paper.

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Correspondence to Günther Schmalzing.

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Haeger, S., Kuzmin, D., Detro-Dassen, S. et al. An intramembrane aromatic network determines pentameric assembly of Cys-loop receptors. Nat Struct Mol Biol 17, 90–98 (2010). https://doi.org/10.1038/nsmb.1721

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