Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding modes for alpha-bungarotoxin and erabutoxin a.
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.
Spura
A
A
Department of Molecular Pharmacology, Division of Biology and Medicine, Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA.
Riel
R U
RU
Freedman
N D
ND
Agrawal
S
S
Seto
C
C
Hawrot
E
E
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002038:Bungarotoxins / Q000378:metabolism*; D003545:Cysteine; D004870:Erabutoxins / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; C038908:erabutoxin A; D004870:Erabutoxins; D003545:Cysteine