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Conformational restriction blocks glutamate receptor desensitization

Nature Structural & Molecular Biology volume 13, pages 1120–1127 (2006)Cite this article

Abstract

Desensitization is a universal feature of ligand-gated ion channels. Using the crystal structure of the GluR2 L483Y mutant channel as a guide, we attempted to build non-desensitizing kainate-subtype glutamate receptors. Success was achieved for GluR5, GluR6 and GluR7 with intermolecular disulfide cross-links but not by engineering the dimer interface. Crystallographic analysis of the GluR6 Y490C L752C dimer revealed relaxation from the active conformation, which functional studies reveal is not sufficient to trigger desensitization. The equivalent non-desensitizing cross-linked GluR2 mutant retained weak sensitivity to a positive allosteric modulator, which had no effect on GluR2 L483Y. These results establish that the active conformation of AMPA and kainate receptors is conserved and further show that their desensitization requires dimer rearrangements, that subtle structural differences account for their diverse functional properties and that the ligand-binding core dimer is a powerful regulator of ion-channel activity.

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Figure 1: Structural basis for desensitization of glutamate receptor ion channels.
Figure 2: Protein engineering in the dimer interface does not generate non-desensitizing kainate receptors.
Figure 3: Ligand-binding domain dimer formation resulting from disulfide bond cross-links.
Figure 4: Non-desensitizing glutamate receptors created by ligand-binding core disulfide bond cross-links.
Figure 5: Modulation of desensitization by disulfide bond cleavage.
Figure 6: Analysis of disulfide bond–cross-linked receptors.

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Acknowledgements

We thank C. Glasser for technical assistance, P. Seeburg (Max Planck Institute for Medical Research, Heidelberg) and S. Heinemann (Salk Institute for Biological Studies) for the gift of the wild-type GluR plasmids, H. Deng for help with cell culture, M. Zhang for help with protein purification and crystallization, H. Jaffe (Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke) for mass spectral analysis, E. Gouaux for sharing results before publication and A. Plested for reading the manuscript. Nucleic acid sequencing was performed by the National Institute of Neurological Disorders and Stroke DNA sequencing facility, Porter Neuroscience Research Center. This work was supported by the Brown foundation (C.R.), Neuroscience Training grant T32 GM008507 (M.C.W.) and the intramural research program of the National Institute of Child Health and Human Development, US National Institutes of Health, Department of Health and Human Services (M.L.M.).

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Authors and Affiliations

  1. Departments of Neuroscience and Molecular and Human Genetics, Baylor College of Medicine, Houston, 77030, Texas, USA

    Matthew C Weston & Christian Rosenmund

  2. Division of Bioengineering and Physical Science, Department of Health and Human Services, Protein Biophysics Resource, Office of Research Services, US National Institutes of Health, Bethesda, 20892, Maryland, USA

    Peter Schuck

  3. Department of Health and Human Services, Laboratory of Cellular and Molecular Neurophysiology, Porter Neuroscience Research Center, National Institute of Child Health and Human Development, US National Institutes of Health, Bethesda, 20892, Maryland, USA

    Alokesh Ghosal & Mark L Mayer

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  1. Matthew C Weston
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Contributions

Electrophysiological experiments were performed by M.C.W., analytical ultracentrifugation by P.S., biochemistry and crystallography by M.L.M., molecular biology and biochemistry by A.G. Data was interpreted and the paper written by M.L.M., C.R. and M.C.W.

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Correspondence to Mark L Mayer.

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Weston, M., Schuck, P., Ghosal, A. et al. Conformational restriction blocks glutamate receptor desensitization. Nat Struct Mol Biol 13, 1120–1127 (2006). https://doi.org/10.1038/nsmb1178

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