The prokaryotic Erwinia chrysanthemi ligand-gated ion channel (ELIC) is competitively inhibited by acetylcholine (Pan et al., 2012). Acetylcholine is the native agonist of the structurally related family of eukaryotic acetylcholine receptors, which like ELIC are pentameric ligand-gated ion channels. To understand the opposite effect upon acetylcholine binding between ELIC and acetylcholine receptors, we used statistical coupling analysis to predict mutations necessary for installing acetylcholine agonism into ELIC. Statistical coupling analysis was performed on the acetylcholine binding protein from Lymnaea stagnalis. This protein is a structural surrogate for the agonist binding domain of acetylcholine receptors, for which a high-resolution structure in complex with acetylcholine is available. Our analysis identified a group of statistically coupled residues that comprises several amino acids previously implicated in acetylcholine agonism of acetylcholine receptors. Mapping these residues onto ELIC revealed 15 residue discrepancies, 4 of which were chosen for initial mutagenesis based upon their proximity to the known agonist binding site. Electrophysiological characterization of ELIC mutants indicates that the potency of the native agonist, cysteamine, is decreased, highlighting the optimized role wild-type residues serve in native agonism. None of the mutants were activated by acetylcholine, however the double mutant A75D/F133W abolished competitive antagonism by acetylcholine, and instead led to acetylcholine dependent potentiation of cysteamine-induced currents. This work demonstrates the ability of statistical coupling analysis to identify functionally important residues in pentameric ligand-gated ion channels and reveals that acetylcholine can be converted from a competitive antagonist into a potentiator, by installing two residues present in acetylcholine receptors.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/42285 |
| Date | 11 June 2021 |
| Creators | Slobodyanyuk, Mykhaylo |
| Contributors | daCosta, Corrie John Bayley, Chica, Roberto |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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