Abstract
We undertook cysteine substitution mutagenesis and fluorophore conjugation at selected residue positions to map sites of ligand binding and changes in solvent exposure of the acetylcholine-binding protein from Lymnaea stagnalis, a nicotinic receptor surrogate. Acrylodan fluorescence emission is highly sensitive to its local environment, and when bound to protein, exhibits changes in both intensity and emission wavelength that are reflected in the degree of solvent exclusion and the effective dielectric constant of the environment of the fluorophore. Hence, cysteine mutants were generated based on the acetylcholine-binding protein crystal structure and predicted ligand binding sites, and fluorescence parameters were assayed on the acrylodan-conjugated proteins. This approach allows one to analyze the environment around the conjugated fluorophore side chain and the changes induced by bound ligand. Introduction of an acrylodan-cysteine conjugate at position 178 yields a large blue shift with alpha-bungarotoxin association, whereas the agonists and alkaloid antagonists induce red shifts reflecting solvent exposure at this position. Such residue-selective changes in fluorescence parameters suggest that certain ligands can induce distinct conformational states of the binding protein, and that mutually exclusive binding results from disparate portals of entry to and orientations of the bound alpha-toxin and smaller acetylcholine congeners at the binding pocket. Labeling at other residue positions around the predicted binding pocket also reveals distinctive spectral changes for alpha-bungarotoxin, agonists, and alkaloid antagonists.
Highlights
The nicotinic acetylcholine receptor1 is the prototypic member of the superfamily of pentameric ligand-gated ion channels (LGIC), that include ␥-aminobutyric acid, glycine, and serotonin (5-HT3) receptors
Cysteine mutants were generated based on the acetylcholine-binding protein crystal structure and predicted ligand binding sites, and fluorescence parameters were assayed on the acrylodanconjugated proteins
In this family of channels, each subunit contains an extracellular domain encompassing the first ϳ210 amino acids followed by four transmembrane ␣-helical segments; ligand binding sites are formed at subunit interfaces on the extracellular side [1, 2]. nicotinic acetylcholine receptor (nAChR) are members of the Cys-loop family of LGICs, so named because of a conserved disulfide linkage in their amino-terminal, extracellular domains
Summary
Ligands and Labeling Reagents—(ϩ)-Epibatidine, gallamine, ␣-bungarotoxin, nicotine, and carbamylcholine were purchased from Sigma (Fig. 1). d-Tubocurarine chloride was purchased from ICN Pharmaceuticals, Inc. In 200-l reaction vessels, AChBP (0.5 nM binding sites) was incubated with increasing concentrations of either 125I-labeled ␣-bungarotoxin or (ϩ)-epibatidine in a solution of 0.1 mg/ml anti-His SPA beads. 125I-labeled ␣-bungarotoxin was held constant at 20 nM and epibatidine was added in variable concentrations. Final ligand concentrations were 2.5 M for epibatidine, gallamine, ␣-bungarotoxin, d-tubocurarine, metocurine, nicotine, and methyllycaconitine, and 25 M for carbamylcholine. For binding site titration experiments, the concentration of binding sites was held constant at 1 M for epibatidine and ␣-bungarotoxin titrations, and 3 M for d-tubocurarine and gallamine. Rates of dissociation of ␣-bungarotoxin were measured by reacting the preformed complex with a large excess of wildtype, unlabeled AChBP to scavenge the dissociated ligand, and observing the time course of the change in fluorescence emission. 125I-␣-bungarotoxin was added in increasing concentrations to AChBP at 0.5 nM in binding sites. Conversions from EC50 to KD were made using Prism version 3.00 (from GraphPad Software, Inc.)
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