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Principles of activation and permeation in an anion-selective Cys-loop receptor

Abstract

Fast inhibitory neurotransmission is essential for nervous system function and is mediated by binding of inhibitory neurotransmitters to receptors of the Cys-loop family embedded in the membranes of neurons. Neurotransmitter binding triggers a conformational change in the receptor, opening an intrinsic chloride channel and thereby dampening neuronal excitability. Here we present the first three-dimensional structure, to our knowledge, of an inhibitory anion-selective Cys-loop receptor, the homopentameric Caenorhabditis elegans glutamate-gated chloride channel α (GluCl), at 3.3 Å resolution. The X-ray structure of the GluCl–Fab complex was determined with the allosteric agonist ivermectin and in additional structures with the endogenous neurotransmitter l-glutamate and the open-channel blocker picrotoxin. Ivermectin, used to treat river blindness, binds in the transmembrane domain of the receptor and stabilizes an open-pore conformation. Glutamate binds in the classical agonist site at subunit interfaces, and picrotoxin directly occludes the pore near its cytosolic base. GluCl provides a framework for understanding mechanisms of fast inhibitory neurotransmission and allosteric modulation of Cys-loop receptors.

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Figure 1: Architecture of the GluCl cryst –Fab complex.
Figure 2: Ivermectin-binding site and atomic interactions.
Figure 3: Glutamate-binding site and specificity.
Figure 4: Ion channel.
Figure 5: Picrotoxin-binding site.
Figure 6: Ion selectivity.

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Acknowledgements

We are grateful to H. Lester for providing the initial GluCl construct, to D. Cawley for monoclonal antibody production, to J. Michel for Fab fragment cloning and sequencing, to C. Alexander and D. C. Dawson for providing Xenopus oocytes, to M. Mayer for advice and equipment related to oocyte experiments, and to L. Vaskalis for help with illustrations. We thank the staff at the Advanced Photon Source beamline 24-ID-C for assistance with X-ray data collection. We are particularly appreciative of discussions with E.G. laboratory members and E. McCleskey. This work was supported by an individual NIH National Research Service Award (F32NS061404) to R.E.H. E.G. is an investigator with the Howard Hughes Medical Institute.

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R.E.H. and E.G. contributed to all aspects of the project.

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Correspondence to Eric Gouaux.

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Atomic coordinates and structure factors have been deposited with the Protein Data Bank under codes 3RHW, 3RIF, 3RI5 and 3RIA for the GluCl–Fab–ivermectin complex alone and with glutamate, with picrotoxin and with iodide, respectively.

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Hibbs, R., Gouaux, E. Principles of activation and permeation in an anion-selective Cys-loop receptor. Nature 474, 54–60 (2011). https://doi.org/10.1038/nature10139

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