eprintid: 1378725 rev_number: 39 eprint_status: archive userid: 608 dir: disk0/01/37/87/25 datestamp: 2012-12-04 20:30:41 lastmod: 2021-10-21 00:01:56 status_changed: 2012-12-04 20:30:41 type: article metadata_visibility: show item_issues_count: 0 creators_name: Zimmermann, I creators_name: Marabelli, A creators_name: Bertozzi, C creators_name: Sivilotti, LG creators_name: Dutzler, R title: Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations. ispublished: pub divisions: UCL divisions: B02 divisions: C08 divisions: D09 divisions: G02 keywords: Acetylcholine, Allosteric Regulation, Amino Acid Sequence, Animals, Barium, Binding Sites, Calcium, Cations, Divalent, Cell Membrane, Cloning, Molecular, Crystallography, X-Ray, Cysteamine, Electrophysiological Phenomena, Escherichia coli, HEK293 Cells, Humans, Ion Channel Gating, Ligand-Gated Ion Channels, Mutagenesis, Site-Directed, Patch-Clamp Techniques, Prokaryotic Cells, Protein Interaction Mapping, Protein Structure, Tertiary, Xenopus laevis, Zinc note: © 2012 Zimmermann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The research leading to these results has received funding from a grant from the Swiss National Science Foundation (grant no. 31003B_141180) to RD and a grant from BBSRC (BB/J005312/1) to LGC. AM is supported by a UCL Impact studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. abstract: The modulation of pentameric ligand-gated ion channels (pLGICs) by divalent cations is believed to play an important role in their regulation in a physiological context. Ions such as calcium or zinc influence the activity of pLGIC neurotransmitter receptors by binding to their extracellular domain and either potentiate or inhibit channel activation. Here we have investigated by electrophysiology and X-ray crystallography the effect of divalent ions on ELIC, a close prokaryotic pLGIC homologue of known structure. We found that divalent cations inhibit the activation of ELIC by the agonist cysteamine, reducing both its potency and, at higher concentrations, its maximum response. Crystal structures of the channel in complex with barium reveal the presence of several distinct binding sites. By mutagenesis we confirmed that the site responsible for divalent inhibition is located at the outer rim of the extracellular domain, at the interface between adjacent subunits but at some distance from the agonist binding region. Here, divalent cations interact with the protein via carboxylate side-chains, and the site is similar in structure to calcium binding sites described in other proteins. There is evidence that other pLGICs may be regulated by divalent ions binding to a similar region, even though the interacting residues are not conserved within the family. Our study provides structural and functional insight into the allosteric regulation of ELIC and is of potential relevance for the entire family. date: 2012-11-20 official_url: http://dx.doi.org/10.1371/journal.pbio.1001429 vfaculties: VFLS oa_status: green language: eng primo: open primo_central: open_green article_type_text: Journal Article, Research Support, Non-U.S. Gov't verified: verified_manual elements_source: PubMed elements_id: 835620 doi: 10.1371/journal.pbio.1001429 pii: PBIOLOGY-D-12-02223 lyricists_name: Sivilotti, Lucia lyricists_id: LGSIV31 full_text_status: public publication: PLoS Biol volume: 10 number: 11 article_number: e1001429 event_location: United States issn: 1554-9173 citation: Zimmermann, I; Marabelli, A; Bertozzi, C; Sivilotti, LG; Dutzler, R; (2012) Inhibition of the prokaryotic pentameric ligand-gated ion channel ELIC by divalent cations. PLoS Biol , 10 (11) , Article e1001429. 10.1371/journal.pbio.1001429 <https://doi.org/10.1371/journal.pbio.1001429>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/1378725/1/1378725.pdf