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J. Biol. Chem., Vol. 276, Issue 4, 2417-2426, January 26, 2001
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From the Department of Neurobiology, Harvard Medical School,
Boston, Massachusetts 02115
Results of affinity-labeling studies and
mutational analyses provide evidence that the agonist binding sites of
the nicotinic acetylcholine receptor (nAChR) are located at the
Contributions of Torpedo Nicotinic Acetylcholine
Receptor
Trp-55 and 
Trp-57 to Agonist and Competitive Antagonist
Function*
and
-
and -
subunit interfaces. For Torpedo nAChR,
photoaffinity-labeling studies with the competitive antagonist
d-[3H]tubocurarine (dTC) identified two
tryptophans, Trp-55 and Trp-57, as the primary sites of
photolabeling in the non- subunits. To characterize the importance
of Trp-55 and Trp-57 to the interactions of agonists and
antagonists, Torpedo nAChRs were expressed in Xenopus oocytes, and equilibrium binding assays and
electrophysiological recordings were used to examine the functional
consequences when either or both tryptophans were mutated to leucine.
Neither substitution altered the equilibrium binding of dTC. However,
the W57L and W55L mutations decreased acetylcholine (ACh) binding
affinity by 20- and 7,000-fold respectively. For the wild-type,
W55L, and W57L nAChRs, the concentration dependence of channel
activation was characterized by Hill coefficients of 1.8, 1.1, and 1.7. For the W55L mutant, dTC binding at the - site acts not as a
competitive antagonist but as a coactivator or partial agonist. These
results establish that interactions with Trp-55 of the
Torpedo nAChR play a crucial role in agonist binding and in
the agonist-induced conformational changes that lead to channel opening.
*
This research was supported in part by United States Public
Health Service Grant NS 19522.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.
A Harvard Mahoney Neuroscience Institute Fellow. Present address:
Millenium Pharmaceuticals, Inc., Cambridge, MA 02139.
§
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.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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