Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Radovan Spurny,Bert Billen,Rebecca J Howard,Marijke Brams,Sarah Debaveye,Kerry L Price,David A Weston,Sergei V Strelkov,Jan Tytgat,Sonia Bertrand,Daniel Bertrand,Sarah C.R Lummis,Chris Ulens

doi:10.1074/jbc.m112.424507

Abstract

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA(A/C) receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7-β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.

Highlights

Pentameric ligand-gated ion channels are modulated by general anesthetics
From concentration-inhibition curves, we calculated IC50 values of 125 Ϯ 10 ␮M for bromoform (n ϭ 4 –10), 162 Ϯ 26 ␮M for chloroform (n ϭ 2–3), 125 Ϯ 5 mM for ethanol (n ϭ 3– 4), and 72 Ϯ 5 for mM bromoethanol (n ϭ 3–9) (Fig. 1c). These results demonstrate that Erwinia chrysanthemi ligand-gated ion channel (ELIC) has pharmacological properties that are different from the related bacterial homolog GLIC and the more distant human Pentameric ligand-gated ion channels (pLGICs)
Similar to nicotinic acetylcholine receptors (nAChRs) [45], GABACRs [4], and GLIC [16, 17], ELIC exhibited inhibition by alcohols and other general anesthetics. These results are consistent with the expanding theme of pLGIC sensitivity to general anesthetic modulation [2] and support the relevance of ELIC as a model for pLGIC function as well as structure

Summary

Background

Results: The crystal structure of ELIC in complex with bromoform reveals anesthetic binding in the channel pore and in novel sites in the transmembrane and extracellular domain. A third site is novel and is located in the extracellular domain in a hydrophobic pocket between the ␤7–␤10 strands Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels. Several specific protein sites in pLGICs have been implicated in general anesthetic binding Channels in this family are pentamers of identical or similar subunits, each of which contributes to a ligand binding extracellular domain, a pore-forming transmembrane domain, and a variable cytoplasmic domain [5]. We sought to extend the observation of general anesthetic modulation of pLGICs to the model system of ELIC, which is a recently discovered GABA-activated bacterial pLGIC [21,22,23,24]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION