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J Biol Chem, Vol. 275, Issue 17, 12651-12660, April 28, 2000
From the Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115
To characterize the structural requirements for
ligand orientation compatible with activation of the
Torpedo nicotinic acetylcholine receptor (nAChR), we used
Cys mutagenesis in conjunction with sulfhydryl-reactive reagents to
tether primary or quaternary amines at defined positions within the
agonist binding site of nAChRs containing mutant
Mapping the Agonist Binding Site of the Nicotinic Acetylcholine
Receptor
ORIENTATION REQUIREMENTS FOR ACTIVATION BY COVALENT AGONIST*
- or 
-subunits
expressed in Xenopus oocytes. 4-(N-Maleimido)benzyltrimethylammonium and
2-aminoethylmethanethiosulfonate acted as irreversible antagonists when
tethered at Y93C, Y198C, or E57C, as well as at N94C
(2-aminoethylmethanethiosulfonate only).
[2-(Trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which
attaches thiocholine to binding site Cys, also acted as an irreversible
antagonist when tethered at Y93C, N94C, or E57C. However,
MTSET modification of Y198C resulted in prolonged activation of the
nAChR not reversible by washing but inhibitable by subsequent exposure
to non-competitive antagonists. Modification of Y198C (or any of the
other positions tested) by
[(trimethylammonium)methyl]methanethiosulfonate resulted only in
irreversible inhibition, while modification of Y198C by
[3-(trimethylammonium)propyl]methanethiosulfonate resulted in
irreversible activation of nAChR, but at lower efficacy than by MTSET.
Thus changing the length of the tethering arm by less than 1 Å in
either direction markedly effects the ability of the covalent
trimethylammonium to activate the nAChR, and agonist activation depends
on a very selective orientation of the quaternary ammonium within the
agonist binding site.
*
This work was supported in part by United States Public
Health Services Grant NS 19522 (to J. B. C.) and by a
Muscular Dystrophy Association Research Development grant (to D. A. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Neurobiology,
Harvard Medical School, 220 Longwood Ave., Boston, MA 02115. Tel.:
617-432-1728; Fax: 617-734-7557; E-mail:
jonathan_cohen@hms.harvard.edu.
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