Probing the Agonist Domain of the Nicotinic Acetylcholine Receptor by Cysteine Scanning Mutagenesis Reveals Residues in Proximity to the 
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- 作者：Armin Spura ; Tanya S. Russin ; Neal D. Freedman ; Marianne Grant ; James T. McLaughlin ; and Edward Hawrot
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：April 20, 1999
- 年：1999
- 卷：38
- 期：16
- 页码：4912 - 4921
- 全文大小：91K
- 年卷期：v.38,no.16(April 20, 1999)
- ISSN：1520-4995
文摘
We have constructed a series of cysteine-substitution mutants in order to identify residues inthe mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in 
-bungarotoxin (-Bgtx)binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of theintroduced cysteines with thiol-specific reagents reveals that subunit residues W187, V188, F189, Y190,and P194 are solvent accessible and are in a position to contribute to the -Bgtx binding site in nativereceptors. These results with the intact receptor are consistent with NMR studies of an -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 between AChRsthat bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C witheither bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET),both methylammonium-containing thiol-modifying reagents with agonist properties, results in a markeddecrease (~55-70%) in the number of -Bgtx binding sites, as measured under saturating conditions.The decrease in sites appears to affect both /
and /
sites to the same extent, as shown for W187Cand F189C which were the two mutants examined on this issue. In contrast to the results obtained withMTSET 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 maximalbinding of -Bgtx to the W187C, V188C, or F189C mutant receptors. The striking alkylammoniumdependency suggests that an interaction of the tethered modifying group with the negative subsite withinthe agonist binding domain is primarily responsible for the observed blockade of toxin binding.
-bungarotoxin (-Bgtx)binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of theintroduced cysteines with thiol-specific reagents reveals that subunit residues W187, V188, F189, Y190,and P194 are solvent accessible and are in a position to contribute to the -Bgtx binding site in nativereceptors. These results with the intact receptor are consistent with NMR studies of an -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 between AChRsthat bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C witheither bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET),both methylammonium-containing thiol-modifying reagents with agonist properties, results in a markeddecrease (~55-70%) in the number of -Bgtx binding sites, as measured under saturating conditions.The decrease in sites appears to affect both / and / sites to the same extent, as shown for W187Cand F189C which were the two mutants examined on this issue. In contrast to the results obtained withMTSET 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 maximalbinding of -Bgtx to the W187C, V188C, or F189C mutant receptors. The striking alkylammoniumdependency suggests that an interaction of the tethered modifying group with the negative subsite withinthe agonist binding domain is primarily responsible for the observed blockade of toxin binding.