Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site

We have constructed a series of cysteine-substitution mutants in order to i dentify residues in the mouse muscle nicotinic acetylcholine receptor (AChR ) that are involved in alpha-bungarotoxin (alpha-Bgtx) binding. Following t ransient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alph a subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the alpha-Bgtx binding site in nativ e receptors. These results with the intact receptor are consistent with NMR studies of an alpha-Bgtx/receptor-dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially be tween AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatmen t of intact cells expressing F189C with either bromoacetylcholine (BrACh) o r [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylam monium-containing thiol-modifying reagents with agonist properties, results in a marked decrease (similar to 55-70%) in the number of alpha-Bgtx bindi ng sites, as measured under saturating conditions. The decrease in sites ap pears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alpha W187C and alpha F189C which were the two mutants examine d on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as meth ylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl meth ane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthios ulfonate (MTSEA), has little or no effect on the maximal binding of alpha-B gtx to the alpha W187C, alpha V188C, or alpha F189C mutant receptors. The s triking alkylammonium dependency suggests that an interaction of the tether ed modifying group with the negative subsite within the agonist binding dom ain is primarily responsible for the observed blockade of toxin binding.