Probing the Agonist Domain of the Nicotinic Acetylcholine Receptor by Cysteine Scanning Mutagenesis Reveals Residues in Proximity to the α-Bungarotoxin Binding Site

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Abstract
We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in α-bungarotoxin (α-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that α subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the α-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an α-Bgtx/receptor−dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry32, 12290−12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease (∼55−70%) in the number of α-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both α/γ and α/δ sites to the same extent, as shown for αW187C and αF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of α-Bgtx to the αW187C, αV188C, or αF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.