Chrysanthemi Ligand-gated Ion Channel Research Articles

Biochemical and Thermodynamic Characterization of the Erwinia Chrysanthemi Ligand-Gated Ion Channel

Biochemical and Thermodynamic Characterization of the Erwinia Chrysanthemi Ligand-Gated Ion Channel

Thermoanalytical Characterization of Pentameric Elic Based on Detergent Size

Differential Scanning Calorimetry has long been used for thermal stability studies of proteins. Erwinia Chrysanthemi Ligand-Gated Ion Channel (ELIC) is a prokaryotic analogue of the nicotinic Acetylcholine Receptor (nAChR), a eukaryotic pentameric Ligand-Gated Ion Channel (pLGIC) responsible for action potential propagation through nerve cells. Elucidation of the melting temperatures (Tm) of the extracellular domain (ECD) and the transmembrane domain (TMD) were achieved using “Intermediate” (C10, C11 and C12) and “Long-Chained” maltoside detergents (C13 and C14). Studies using the “Intermediate” maltosides suggest increased stability by over 8°C to the hydrophobic TMD without altering the hydrophilic ECD's stability. There is an obvious correlation between increased length of the detergent and increased stability of the TMD. However, “Long-Chained” maltosides appear to provide such a large increase in thermal stability of the TMD (>15°C) that it is conveyed to the ECD, as well, resulting in an increase of over 4°C. Thus, by manipulating the detergent length we were able to separate and identify the Tm's of the TMD and ECD. As well, we have provided novel stability data for improving crystal structures and other detergent-based experimentation. AHA- 11SDG5440003, NIH 1RO1GM097159-01A1 and Welch Foundation BI-1757.

Insights into the binding of GABA to the insect RDL receptor from atomistic simulations: a comparison of models

The resistance to dieldrin (RDL) receptor is an insect pentameric ligand-gated ion channel (pLGIC). It is activated by the neurotransmitter γ-aminobutyric acid (GABA) binding to its extracellular domain; hence elucidating the atomistic details of this interaction is important for understanding how the RDL receptor functions. As no high resolution structures are currently available, we built homology models of the extracellular domain of the RDL receptor using different templates, including the widely used acetylcholine binding protein and two pLGICs, the Erwinia Chrysanthemi ligand-gated ion channel (ELIC) and the more recently resolved GluCl. We then docked GABA into the selected three dimensional structures, which we used as starting points for classical molecular dynamics simulations. This allowed us to analyze in detail the behavior of GABA in the binding sites, including the hydrogen bond and cation-π interaction networks it formed, the conformers it visited and the possible role of water molecules in mediating the interactions; we also estimated the binding free energies. The models were all stable and showed common features, including interactions consistent with experimental data and similar to other pLGICs; differences could be attributed to the quality of the models, which increases with increasing sequence identity, and the use of a pLGIC template. We supplemented the molecular dynamics information with metadynamics, a rare event method, by exploring the free energy landscape of GABA binding to the RDL receptor. Overall, we show that the GluCl template provided the best models. GABA forming direct salt-bridges with Arg211 and Glu204, and cation-π interactions with an aromatic cage including Tyr109, Phe206 and Tyr254, represents a favorable binding arrangement, and the interaction with Glu204 can also be mediated by a water molecule.

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

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.

Structure and Function of ELIC Bound with the Antagonist Acetylcholine

The bacterium Erwinia chrysanthemi ligand gated ion channel (ELIC) is a prototype for Cys-loop receptors, including nicotinic acetylcholine receptors (nAChRs). We co-crystallized ELIC with acetylcholine, an endogenous agonist of nAChRs, and solved the crystal structure to a resolution of 2.9 A. Acetylcholine binds to the orthosteric agonist site for Cys-loop receptors. Upon acetylcholine binding, loop C exhibits a substantial contraction with a profound reduction in flexibility. Conformational changes in nearby regions of the ligand-binding domain are also observed. However, no substantial reorganization in the pore region was identified except for subtle side chain reorientation of the pore constriction residue F247. Functional measurements on Xenopus oocytes expressing ELIC channels indicate that acetylcholine antagonizes the ELIC currents elicited by primary amines. Our high-resolution structure provides an atomic-level detailed picture of how ligand binding renders gating-related conformational changes in different domains of Cys-loop receptors. It also represents a valuable template for rational drug development. (Supported by NIH grants R01GM066358, R01GM056257, R37GM049202, and T32GM075770)