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J. Biol. Chem., Vol. 275, Issue 38, 29441-29451, September 22, 2000
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From the Most general anesthetics including long chain
aliphatic alcohols act as noncompetitive antagonists of the nicotinic
acetylcholine receptor (nAChR). To locate the sites of
interaction of a long chain alcohol with the Torpedo nAChR,
we have used the photoactivatible alcohol
3-[3H]azioctanol, which inhibits the nAChR and
photoincorporates into nAChR subunits. At 1 and 275 µM,
3-[3H]azioctanol photoincorporated into nAChR subunits
with increased incorporation in the
Identification of Sites of Incorporation in the Nicotinic
Acetylcholine Receptor of a Photoactivatible General Anesthetic*
,¶
Department of Neurobiology, Harvard Medical
School, Boston, Massachusetts 02115 and the § Department of
Anesthesia and Critical Care, Massachusetts General Hospital, Boston,
Massachusetts 02114 and Department of Biological Chemistry
and Molecular Pharmacology, Harvard Medical School, Boston,
Massachusetts 02115
-subunit in the desensitized
state. The incorporation into the -subunit was mapped to two large
proteolytic fragments. One fragment of ~20 kDa (V8-20), containing
the M1, M2, and M3 transmembrane segments, showed enhanced
incorporation in the presence of agonist whereas the other of ~10 kDa
(V8-10), containing the M4 transmembrane segment, did not show
agonist-induced incorporation of label. Within V8-20, the primary
site of incorporation was Glu-262 at the C-terminal end of
M2, labeled preferentially in the desensitized state. The
incorporation at Glu-262 approached saturation between 1 µM, with ~6% labeled, and 275 µM, with
~30% labeled. Low level incorporation was seen in residues at the
agonist binding site and the protein-lipid interface at ~1% of the
levels in Glu-262. Therefore, the primary binding site of
3-azioctanol is within the ion channel with additional lower affinity
interactions within the agonist binding site and at the protein-lipid interface.
*
This work was supported by United States Public Health
Service Grant GM 58448 and by an award in structural neurobiology from the Keck Foundation.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.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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