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1.
J Biol Chem ; 275(29): 22452-60, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10791957

ABSTRACT

Although previous results indicate that alpha-subunit residues Trp(187), Val(188), Phe(189), Tyr(190), and Pro(194) of the mouse nicotinic acetylcholine receptor are solvent-accessible and are in a position to contribute to the alpha-bungarotoxin (alpha-Bgtx) binding site (Spura, A., Russin, T. S., Freedman, N. D., Grant, M., McLaughlin, J. T., and Hawrot, E. (1999) Biochemistry 38, 4912-4921), little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at alpha184-alpha197 with the thiol-specific biotin derivative (+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine , we now show that only very subtle differences in reactivity (approximately 10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin alpha-Bgtx is significantly retarded for positions alphaW187C, alphaF189C, and reduced wild-type receptors (alphaCys(192) and alphaCys(193)), further emphasizing their major contribution to the alpha-Bgtx binding site. Interestingly, although biotinylation of position alphaV188C is not affected by the presence of alpha-Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position alphaV188C, but not at alphaW187C or alphaF189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor.


Subject(s)
Bungarotoxins/metabolism , Erabutoxins/metabolism , Receptors, Nicotinic/metabolism , Amino Acid Substitution , Animals , Binding Sites/genetics , Cysteine , Mice , Protein Binding , Receptors, Nicotinic/genetics , Signal Transduction
2.
Biochemistry ; 38(16): 4912-21, 1999 Apr 20.
Article in English | MEDLINE | ID: mdl-10213592

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 alpha-bungarotoxin (alpha-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alpha 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 native 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 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 ( approximately 55-70%) in the number of alpha-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alphaW187C and alphaF189C 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 alpha-Bgtx to the alphaW187C, alphaV188C, or alphaF189C 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.


Subject(s)
Bungarotoxins/metabolism , Cysteine/genetics , Nicotinic Agonists/chemistry , Receptors, Nicotinic/chemistry , Acetylcholine/analogs & derivatives , Acetylcholine/chemistry , Animals , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/genetics , Humans , Indicators and Reagents , Mesylates/chemistry , Mice , Mutagenesis, Site-Directed , Nicotinic Agonists/metabolism , Nicotinic Antagonists/chemistry , Nicotinic Antagonists/metabolism , Oxidation-Reduction , Peptide Fragments/genetics , Phenylalanine/genetics , Protein Binding/genetics , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Torpedo , Tryptophan/genetics , Valine/genetics
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