Identification of Sites of Incorporation in the Nicotinic Acetylcholine Receptor of a Photoactivatible General Anesthetic*

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 α-subunit in the desensitized state. The incorporation into the α-subunit was mapped to two large proteolytic fragments. One fragment of ∼20 kDa (αV8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of ∼10 kDa (αV8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within αV8-20, the primary site of incorporation was αGlu-262 at the C-terminal end of αM2, labeled preferentially in the desensitized state. The incorporation at αGlu-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 αGlu-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.

The molecular sites of actions of general anesthetics are currently unknown. However, in recent years, evidence for the direct interaction between general anesthetics and specific proteins has accumulated (1). In particular, at clinically effective concentrations most volatile general anesthetics perturb ligand-gated ion channels. For example, they enhance agonist action on inhibitory receptors including most GABA A 1 and glycine receptors. They also noncompetitively inhibit excitatory receptors such as nicotinic acetylcholine receptors (nAChR) and serotonin 5HT 3 receptor. These ion channels belong to a superfamily that is composed of five homologous subunits arranged as a pseudopentamer with each subunit composed of a large N-terminal extracellular segment and four transmembrane segments, M1-M4. The two agonist binding sites are located in the N-terminal extracellular segment at subunit interfaces. Based on photoaffinity labeling and mutational analyses of the nAChR, the M2 segments of each subunit are ␣-helices arranged around the central axis and contribute to the lumen of the nAChR ion channel. Additionally, several nAChR noncompetitive antagonists have been shown to bind within the lumen of the ion channel (2)(3)(4).
Although evidence exists for direct binding of anesthetics on ligand-gated ion channels, the binding sites have not been clearly located. In the GABA A and glycine receptors, mutational analyses have identified two residues, one each in the transmembrane segments M2 and M3, which modulate the action of a variety of general anesthetics, including long chain alcohols (5)(6)(7). These residues have been hypothesized to contribute to an anesthetic site (8), but other groups (1,9,10) have suggested that these and neighboring residues act allosterically on anesthetic sites elsewhere in the receptor.
In muscle nAChR, single channel studies with long chain alcohols and other anesthetics, such as isoflurane, suggest that these anesthetics bind within the ion channel. The open channel state in the presence of these drugs is characterized by flickering, similar to that seen with QX-222, an aromatic amine channel blocker (11). However, butanol and hexanol do not compete with QX-222 for a common binding site (12). In flux studies with nAChR-rich membranes from Torpedo electric organ, octanol and heptanol do compete with each other but not with procaine (13). Site-directed mutagenesis of muscle nAChR has shown that the nature of the residue at the M2 position 10Ј (based on numbering from the conserved positive charge at the N terminus of M2), facing the lumen of the ion channel, can increase the potency of long chain alcohols and isoflurane as channel blockers (14).
Because of the difficulty of interpreting mutational studies in this highly allosteric family of receptors, a complementary approach, photoaffinity labeling, is attractive. The photoaffinity general anesthetic 3-azioctanol was developed (15) as a probe of the binding sites of long chain alcohols. This compound acts as an anesthetic in tadpoles, producing a loss of righting reflex with an EC 50 of ϳ160 M, an EC 50 that is about one-third of the potency of octanol. For the GABA A receptor, 3-azioctanol potentiates the response to submaximal concentrations of GABA, and it inhibits agonist activation of muscle-type nAChR. Additionally, 1 M 3-[ 3 H]azioctanol was shown to photoincorporate into subunits of the Torpedo nAChR with preferential incorporation into the ␣-subunit in the presence of agonist. This agonist-dependent incorporation was localized to a 20-kDa fragment containing the first three transmembrane fragments.
In the present work, 3-[ 3 H]azioctanol has been used as a photoaffinity probe to localize further the sites of interaction of a long chain alcohol with Torpedo nAChR-rich membranes. The primary site of incorporation in the presence of agonist was mapped to ␣Glu-262, at the C terminus of ␣M2. Additional sites of incorporation were found although at lower efficiency than the incorporation at ␣Glu-262. The levels of incorporation at 1 and 275 M indicated that the incorporation at ␣Glu-262 approached saturation across this concentration range, whereas the incorporation at the other sites increased linearly. Therefore, ␣Glu-262 is within the high affinity binding site of long chain alcohol anesthetics.

EXPERIMENTAL PROCEDURES
Materials-nAChR-enriched membranes were isolated from Torpedo californica electric organ (16). The final membrane suspensions were stored in 38% sucrose at Ϫ80°C under argon. The membranes used here contained 0.5-2.0 nmol of acetylcholine binding sites per milligram of protein. 3-[ 3 H]Azioctanol and nonradioactive 3-azioctanol were synthesized as described previously (15). The specific activity of the 3-[ 3 H]azioctanol was ϳ11 Ci/mmol. This stock was stored at Ϫ20°C in CH 2 Cl 2 , which was removed via evaporation immediately prior to the addition of membranes or isotopic dilution. For studies of incorporation at concentrations higher than 1 M 3-[ 3 H]azioctanol, this stock was isotopically diluted with a stock of nonradioactive 3-azioctanol, 11 mM (concentration determined by the absorbance of 3-azioctanol at 350 nm (15)) in Torpedo physiological saline (TPS: 250 mM NaCl, 5 mM KCl, 3 mM CaCl 2 , 2 mM MgCl 2 , 5 mM sodium phosphate, pH 7.0), to a final specific activity of ϳ0.04 Ci/mmol. This dilution was prepared immediately before addition to membranes. Staphylococcus aureus glutamylendopeptidase (V8 protease) was from ICN Biomedical Inc, endoproteinase Lys C (EndoLysC) from Roche Molecular Biochemicals, and phencyclidine from Alltech Associates. Gallamine triethyl iodide was from Lederle, phenyltrimethylammonium from Aldrich, and trifluoroacetic acid was from Pierce. 1-Azidopyrene (1-AP) was purchased from Molecular Probes. 10% Genapol C-100 was from Calbiochem. Nicotine, d-tubocurarine, and carbamylcholine were from Sigma. Pancuronium was from Organon; ␣-bungarotoxin (␣BgTx) was purchased from Biotoxins, Inc.
Photoaffinity Labeling of nAChR-enriched Membranes with 3-[ 3 H] Azioctanol-For analytical labeling experiments, freshly thawed Torpedo membranes were diluted with TPS and pelleted (15000 ϫ g) for 30 min, and the pellets were resuspended in TPS at 2 mg/ml protein (ϳ1 M nAChR). Membrane aliquots (100 g per condition) were combined with 3-[ 3 H]azioctanol in the absence or presence of other ligands as noted in the figure legends. When one of the conditions contained ␣BgTx, the samples were incubated for 2 h in the dark at room temperature in glass vials; otherwise, the samples were irradiated within 3 min of the addition of drugs. The suspensions were irradiated at 365 nm (Spectroline lamp EN-16) for 10 min in a plastic 96-well plate on ice. The incorporation in the presence of carbamylcholine after 10 min of photolysis was near the half-maximal incorporation in nAChR ␣-subunit, with the maximum near 40 min of photolysis. The incorporation in the ␣-subunit in the presence of carbamylcholine without irradiation was 4% of the levels seen following 10 min irradiation. For the remainder of the experiments, the photolysis was carried out for 10 min. The suspensions were diluted with sample loading buffer and directly submitted to SDS-PAGE.
For proteolytic mapping of 3-[ 3 H]azioctanol-labeled ␣-subunit with S. aureus V8 protease (17,18), labeling was carried out with 400 g (analytical mapping) or 10 mg (preparative) nAChR-rich Torpedo membranes. For analytical mapping, samples were photolyzed in a 24-well plate, whereas for preparative mapping the samples were photolyzed in glass screw-top vials with a stir bar. Following photolysis, the membrane suspensions were pelleted as described above. For analytical mapping, samples were resuspended in sample buffer and submitted to SDS-PAGE. For preparative mapping, samples were resuspended at 2 mg/ml in TPS. The samples were labeled further with 1-AP (19) to ease identification and isolation of subunits and fragments from gels. 1-AP (62.5 mM in Me 2 SO) was added to a final concentration of 500 M. After a 90-min incubation, the samples were photolyzed for 15 min on ice using a 365 nm lamp (Spectroline EN-16). Membranes were pelleted (15,000 ϫ g) for 30 min, resuspended in sample buffer, and submitted to SDS-PAGE.
Gel Electrophoresis-SDS-PAGE was performed as described by Laemmli (20), with modifications (18). For analytical gels, the polypeptides were resolved on a 1-mm thick 8% acrylamide gel and visualized by staining with Coomassie Blue (0.25% w/v in 45% methanol and 10% acetic acid). For autoradiography, the gels were impregnated with fluor (Amplify, Amersham Pharmacia Biotech), dried, and exposed at Ϫ80°C to Eastman Kodak X-OMAT film for various times (6 -8 weeks). Additionally, incorporation of 3 H into individual polypeptides was quantified by scintillation counting of excised gel slices (21). For analytical V8mapping gels, following electrophoresis, the gels were briefly stained with Coomassie Blue and destained to allow visualization of the subunits. The ␣-subunits were then excised and placed directly into individual wells of a 1.5-mm mapping gel, composed of a 5-cm 4.5% acrylamide stacking gel, and a 15-cm 15% acrylamide separating gel. Into each well was added 1:1 gram subunit:gram S. aureus V8 protease in overlay buffer (5% sucrose, 125 mM Tris-HCl, 0.1% SDS, pH 6.8). The gel was run at 150 V for 2 h, and then the current was turned off for 1 h. The gel was then run at constant current overnight until the dye front reached the end of the gel. The gel was stained, and 3 H was quantified by liquid scintillation counting. For preparative labeling, the polypeptides were resolved on a 1.5-mm thick 8% acrylamide gel. The ␣-subunit was identified in 8% gels by 1-AP fluorescence and then excised and loaded directly onto the 1.5-mm mapping gels. The ␣-subunit proteolytic fragments of ϳ20 kDa (␣V8-20) and ϳ10 kDa (␣V8-10) were identified by fluorescence and excised. The region between ␣V8-20 and ␣V8-10 was excised to isolate ␣V8-18. The excised proteolytic fragments were isolated by passive elution into 0.1 M NH 4 HCO 3 , 0.1% SDS (19). The eluate was filtered (Whatman No. 1) and concentrated using Millipore M r 5,000 concentrators. To remove excess SDS, acetone was added to the concentrate. Following incubation at Ϫ20°C overnight, the peptides were pelleted.
Proteolytic Digestion-For EndoLysC digestion, acetone-precipitated subunits or subunit fragments were resuspended in 15 mM Tris, pH 8.1, 0.1% SDS. EndoLysC (1.5 milliunit in resuspension buffer) was added to a final volume of 100 l. The digestion was allowed to proceed for 7-9 days before separation of fragments by HPLC. For S. aureus V8 protease digestion in solution, acetone-precipitated peptides were resuspended in 15 mM Tris, pH 8.1, 0.1% SDS. V8 protease in resuspension buffer was added to a final concentration of 1:1 (w/w) and incubated at room temperature for 3-4 days before separation of fragments by HPLC. For trypsin digestion, acetone-precipitated peptides were resuspended in a small volume (40 l) of 100 mM NH 4 HCO 3 , 0.1% SDS, pH 7.8. Genapol C-100 and trypsin were added to a final concentration of 0.02% SDS, 0.5% Genapol C-100, and 1:1 (w/w) trypsin. The digestion was allowed to proceed 3-4 days at room temperature prior to separation of the fragments by HPLC.
HPLC Purification-Proteolytic fragments from enzymatic digestion of ␣V8-20 and ␣V8-10 fragments labeled with 3-[ 3 H]azioctanol were further purified by reverse-phase HPLC (22), using a Brownlee C4-Aquapore column (100 ϫ 2.1 mm, 7-m particle size). Solvent A was 0.08% trifluoroacetic acid in water, and solvent B was 0.05% trifluoroacetic acid in 60% acetonitrile, 40% 2-propanol. A nonlinear gradient (Waters Model 680 gradient controller, curve No. 7) from 25 to 100% solvent B in 80 min was used. The rate of flow was 0.2 ml/min, and 0.5-ml fractions were collected. The elution of peptides was monitored by absorbance at 215 nm, and the fluorescence from 1-AP was detected by fluorescence emission (357-nm excitation, 432-nm emission). Additionally, aliquots from the fractions were taken to determine the distribution of 3 H by liquid scintillation counting.
Sequence Analysis-Automated N-terminal sequence analysis was performed on an Applied Biosystems Model 477A protein sequencer with an in-line 120A PTH analyzer. HPLC samples (450-l fractions) were directly loaded onto chemically modified glass fiber disks (Beckman) in 20-l aliquots, allowing the solvent to evaporate at 40°C between loads. Sequencing was performed using gas-phase trifluoroacetic acid to minimize possible hydrolysis. After conversion of the released amino acids to PTH-amino acids, the suspension was divided into two parts. One portion, approximately one-third, went to the PTH analyzer whereas the remaining two-thirds were collected for scintillation counting. Yield of PTH-amino acids was calculated from peak height compared with standards using the Model 610A Data Analysis Program Version 1.2.1. Initial and repetitive yields were calculated by a nonlinear least squares regression to the equation M ϭ I 0 ϫ R n , where M is the observed release, I 0 is the initial yield, R is the repetitive yield, and n is the cycle number. PTH-derivatives known to have poor recovery (Ser, Arg, Cys, and His) were omitted from the fit.
Incorporation of radioactivity in fragments and residues was quantified based on the results of sequence analysis. For the ␣V8-20 and ␣V8-10 fragments, approximately equal aliquots were subjected to either liquid scintillation counting or sequence analysis. Prior to sequencing, these samples (because they contained SDS) were treated on the sequencing filters for 4 min with gas-phase trifluoroacetic acid, followed by a 5-min wash with ethyl acetate. To estimate incorporation in large subunit fragments (␣V8-20 and ␣V8-10) or in fragments isolated by HPLC, incorporation was calculated as the 3 H loaded divided by three times the observed initial yield of the sequence (three times because only one-third of the PTH-amino acids was measured for mass calculations). Because even under favorable conditions less than 50% of the material loaded is capable of being sequenced, this calculation is an overestimate. For incorporation at specific residues, the mass of that residue was calculated from the initial and repetitive yields. The increased 3 H release in that cycle (cpm n -cpm n-1 ) was divided by twice the mass of that cycle (twice because 2-fold more PTH-amino acids were assayed for 3 H than for mass). In this calculation, the radioactivity released and the mass levels reflect only the sequenced material.  (24). The 3 H incorporation in VDAC, although not affected by the presence of carbamylcholine, was reduced by 50% at the higher concentration of 3-[ 3 H]azioctanol. This decrease suggests specific incorporation of 3-[ 3 H]azioctanol in VDAC, which is inhibited by excess non-radioactive 3-azioctanol. The 3 H incorporation in other non-receptor polypeptides (rapsyn (43 kDa) and the ␣-subunit of (Na ϩ /K ϩ )-ATPase (␣NK)) was not altered by the presence of carbamylcholine and appeared similar at 1 and 275 M 3-[ 3 H]azioctanol.

Photoincorporation of 3-[ 3 H]Azioctanol into nAChR-rich
Of the nAChR subunits, ␣ was labeled most strongly. Incorporation of 3-[ 3 H]azioctanol into the ␣-subunit was dependent on the conformational state of the nAChR, as the presence of agonist resulted in enhanced incorporation into the ␣-subunit but not in non-nAChR polypeptides. Based on scintillation counting of excised gel slices, the increase in incorporation was on average ϳ5-fold at 1 M 3- The enhancement of 3-[ 3 H]azioctanol photolabeling in the ␣-subunit by carbamylcholine as well as other cholinergic agonists and competitive antagonists ( Fig. 2) was determined by quantification of 3 H incorporation in gel slices. At concentrations sufficient to fully occupy the ACh site, the agonists phenyltrimethylammonium and nicotine increased 3-[ 3 H]azioctanol photoincorporation in the ␣-subunit to the same extent as carbamylcholine. The presence of the competitive antagonists d-tubocurarine and gallamine, known to partially desensitize the receptor (25,26), resulted in incorporation in the ␣-subunit ϳ60% of that seen in the presence of carbamylcholine, whereas for pancuronium, a competitive antagonist that is not known to desensitize the receptor, 3-[ 3 H]azioctanol incorporation into the ␣-subunit was similar to that seen in the absence of carbamylcholine. No effect of these cholinergic drugs was seen on the incorporation of 3-[ 3 H]azioctanol into non-nAChR polypeptides including rapsyn (43 kDa), calelectrin (37 kDa), or the (Na ϩ / K ϩ )-ATPase ␣-subunit (data not shown). The dependence of nAChR ␣-subunit-labeling upon carbamylcholine concentration indicated that the enhanced labeling was a property of the desensitized state of the nAChR. Incorporation as a function of carbamylcholine concentration was well fit by a single site model, with a K ϭ 4 M (data not shown). Whereas this value for the concentration of carbamylcholine producing 50% enhancement was higher than the directly measured K eq of 0.1 M (27), this discrepancy was not unexpected because the photolabeling experiment was carried out at a concentration of ACh sites of 2.2 M.
The effects of several noncompetitive antagonists on the incorporation of 3-[ 3 H]azioctanol at 1 M were also tested (Fig.  3A). For membranes equilibrated with carbamylcholine, the 3 H incorporation in the nAChR ␣-subunit was insensitive to the presence of 1 mM octanol. At 100 M, meproadifen, an aromatic amine noncompetitive antagonist, reduced the incorporation by ϳ50%. Two other aromatic amine noncompetitive antagonists, phencyclidine and QX-222, failed to inhibit the incorporation of 3-[ 3 H]azioctanol in the ␣-subunit (data not shown). The presence of these noncompetitive antagonists did not affect the incorporation in the other nAChR subunits (data not shown) nor the incorporation in non-nAChR polypeptides including rapsyn (43 kDa), VDAC (34 kDa), and calectrin (37 kDa) (data not shown).
The effects of meproadifen were also studied in the presence of 275 M 3-[ 3 H]azioctanol (Fig. 3B). At that concentration the presence of carbamylcholine resulted in an ϳ3-fold increase in the incorporation of 3-[ 3 H]azioctanol in the ␣-subunit over that seen in the absence of carbamylcholine. In the presence of carbamylcholine, meproadifen reduced the incorporation by ϳ50%. In the absence of carbamylcholine, meproadifen actually enhanced the 3-[ 3 H]azioctanol incorporation. In the presence of ␣BgTx, meproadifen did not alter the 3-[ 3 H]azioctanol incorporation in the ␣-subunit. The incorporation in rapsyn (43 kDa) was not affected by the presence of these cholinergic drugs.
The incorporation of 3-[ 3 H]azioctanol in nAChR ␣-subunit was measured over a range of 3-[ 3 H]azioctanol concentrations, using a constant specific activity of 3-[ 3 H]azioctanol (Fig. 4). The incorporation in the (Na ϩ /K ϩ )-ATPase ␣-subunit (open symbols) increased linearly across the range of concentrations tested and was not affected by the presence of cholinergic drugs. For membranes equilibrated with carbamylcholine, the incorporation in the ␣-subunit increased up to ϳ1 mM and then appeared to saturate. At all concentrations, the incorporation in the presence of ␣BgTx was less than that seen in the absence of added drugs although at ϳ2 mM the incorporation in the presence of ␣BgTx was similar to that seen in the presence of carbamylcholine. In the absence of drug, the incorporation appeared to increase nearly linearly up to 1 mM, and then the incorporation increased sharply, surpassing the incorporation in the presence of carbamylcholine at 2 mM. However, the Based on liquid scintillation counting of these ␣-subunit proteolytic fragments, the main sites of photoincorporation in the absence of agonist were within the ␣V8-20 3 and ␣V8-10 fragments (Fig. 5). The 3 H incorporation in each fragment was similar at both concentrations of 3-[ 3 H]azioctanol. In the absence of agonist, the incorporation in ␣V8-10 was ϳ60% that of ␣V8-20. The addition of agonist increased the labeling of the ␣V8-20 fragment, 9-

3-[ 3 H]Azioctanol
Photoincorporation within the ␣M2 Segment-To determine whether there was incorporation in the ␣M2 segment, the eluted ␣V8-20 fragment, labeled with 3-[ 3 H]azioctanol, was digested with EndoLysC. Digestion with EndoLysC is known to create an ϳ10-kDa fragment starting at ␣Met-243, the N terminus of the ␣M2 segment, that can be purified by reverse-phase HPLC (16). When the EndoLysCdigested ␣V8-20, which had been labeled with 275 M 3-[ 3 H] azioctanol in the presence of carbamylcholine, was fractionated by reverse-phase HPLC, ϳ80% of the 3 H eluted in a peak centered at fraction 33 (ϳ88% organic) (Fig. 6A). For the samples labeled in the presence of ␣BgTx or the absence of other drugs, the 3 H in fraction 33 was only ϳ20% that seen for the sample labeled in the presence of carbamylcholine.
For each labeling condition, fraction 33, which contained the peak of 3 H from the sample labeled in the presence of carbamylcholine, was subjected to Edman degradation (Fig. 6B) showing that the only sequence present was that beginning at ␣Met-243 (Ϫcarb: I 0 ϭ 23 pmol; ϩcarb: I 0 ϭ 30 pmol). No other sequences were present at more than 10% the mass of the 3 Staining of the mapping gel with Coomassie Blue revealed the four expected proteolytic fragments under all conditions. Additionally, a band was present at ϳ22 kDa under the condition labeled with 275 M 3-[ 3 H]azioctanol in the presence of carbamylcholine (data not shown). The band was excised, and moreover, the 22-kDa region was also excised in other conditions. This band was assumed to be ␣V8-20 that was highly labeled with 3-[ 3 H]azioctanol, resulting in ⅐reduced mobility. Therefore, the 3 H present in this region was attributed to labeling in ␣V8-20 and was added to that of the ␣V8-20 band.

FIG. 5. Mapping of sites of 3-[ 3 H]azioctanol incorporation into
the nAChR ␣-subunit using S. aureus V8 protease. nAChR-rich membranes (400 g at 2 mg/ml) were labeled with 1 M (11 Ci/mmol) or 275 M (0.04 Ci/mmol) 3-[ 3 H]azioctanol in the absence or presence of 2 mM carbamylcholine. After photolysis at 365 nm for 10 min, membranes were pelleted, resuspended in sample buffer, and submitted to SDS-PAGE. Following electrophoresis, the ␣-subunit was excised and transferred to the well of a 15% mapping gel for digestion with V8 protease. Bands were visualized with Coomassie Blue, and 3 H incorporation was quantified by scintillation counting. 3

H present in proteolytic fragments of nAChR ␣-subunit labeled in the absence (solid and vertical hatched bars) or presence of 2 mM carbamylcholine at 1 M (solid and open bars) and 275 M (vertical and horizontal hatched bars) 3-[ 3 H]azioctanol is
shown. Inset, the nAChR ␣-subunit proteolytic fragments produced by digestion by V8 protease. peptide beginning at ␣Met-243. For the sample labeled in the presence of carbamylcholine, there was a peak of 3 H release in cycle 20, corresponding to incorporation at ␣Glu-262, and that release was reduced by ϳ60% in the sample labeled in the absence of carbamylcholine or in the presence of ␣BgTx (data not shown). Based upon the 3

3-[ 3 H]Azioctanol Photoincorporation within the ␣M1 and ␣M3 Segments-When
EndoLysC cleaves ␣V8-20 at ␣Lys-242, before ␣M2, it can also cleave the fragment between the N terminus of ␣V8-20 and ␣Lys-242. There are two lysines in this fragment, ␣Lys-179 and ␣Lys-185. Cleavage at either of these two sites will generate a fragment that contains a portion of the ACh binding site (␣-(190 -200)) as well as the ␣M1 segment. This fragment can be resolved from the fragment containing ␣M2 by HPLC purification. The fragment containing ␣M1 elutes in a peak of absorbance and fluorescence near fraction 29 (69% organic) (Fig. 6A). Sequence analysis of this fraction from the sample labeled at 275 M 3-[ 3 H]azioctanol in the presence of carbamylcholine indicated the presence of the sequence beginning at ␣His-186 (I o ϭ 36 pmol) (see Fig. 6C), containing ϳ0.05 mol of incorporated label per mol of fragment. This incorporation is ϳ4% that in the major radiolabeled fragment, recovered in fraction 33, which contained the ␣M2 and ␣M3 segments. Therefore, if there is any incorporation within the ␣M1 segment, it was less than 4% of the level of the incorporation in the ␣M2 segment.

3-[ 3 H]Azioctanol Photoincorporation within the Agonist Binding
Site-For the ␣V8-20 fragment isolated from nAChRs labeled with either 1 or 275 M 3-[ 3 H]azioctanol in the absence of carbamylcholine, the HPLC chromatogram of the EndoLysC digest of ␣V8-20 (Fig. 6A) contained a peak of 3 H at fraction 29 (69% organic) in addition to the peak at fraction 33. When the material in fraction 29 was sequenced, the primary sequence began at ␣His-186 (Ϫcarb: I 0 ϭ 35 pmol; ϩ␣BgTx: I 0 ϭ 55 pmol; ϩcarb: I 0 ϭ 36 pmol) (Fig. 6C). This fragment contains residues contributing to the ACh site (␣-(190 -200)) as well as the ␣M1 segment, because there is no lysine between ␣His-186 and ␣Lys-242 prior to ␣M2. At 275 M 3-[ 3 H]azioctanol, 3 H release was evident in cycles 5 and 13 for the fragment labeled in the absence of carbamylcholine but not for the samples labeled in the presence of carbamylcholine or ␣BgTx. Release of 3 H in these cycles correspond to ␣Tyr-190 and ␣Tyr-198, residues known to contribute to the agonist binding site (2). The amount of incorporation in these residues was ϳ10% that in ␣Glu-262 in the absence of carbamylcholine, with 3-  Table I under "Discussion"). In the presence of carbamylcholine, whereas there was no release in cycle 5 or 13, there was release evident in cycle 3, which, if originating from the fragment beginning at ␣His-186, indicated ϳ0.003 mol incorporated per mol of ␣Val-189.
Additional incorporation is also present within ␣V8-18, though at an undetermined site(s). EndoLysC digestion of ␣V8-18 followed by HPLC separation showed a peak of 3 H that contained a single sequence, that beginning at Lys-77 (Ϫcarb: I o ϭ 22 pmol, ϩ␣BgTx I o ϭ 15 pmol, ϩcarb: I o ϭ 13 pmol) (data not shown). The fragment showed ϳ10% incorporation in each of the three conditions. The incorporation in this fragment

H]azioctanol into fragments and residues of the ␣-subunit
The ratio of moles 3-[ 3 H]azioctanol incorporated per mol of the fragments or residues labeled was calculated from the 3 H incorporation and the known specific activity of 3-[ 3 H]azioctanol. The 3 H incorporation in each fragment and residue was calculated as described under "Experimental Procedures." For the incorporation into fragments, the mass levels were based on the observed mass sequenced and the total radioactivity loaded. For the incorporation into specific residues, the mass was based on the initial and repetitive yields, and the radioactivity was based on the observed release. Averages shown are from duplicate preparative-labeling experiments. accounts for most of the incorporation in ␣V8-18 because there was ϳ6% incorporation in ␣V8-18. Because the radioactive release in the cycle containing ␣Tyr-93, a residue contributing to the agonist binding site, was at background levels, this position was labeled by no more than 0.00003 mol of 3-[ 3 H] azioctanol per mol of residue (data not shown).  (Fig. 9A, inset), whereas only ϳ20% eluted in a broad peak between fractions 32-35 where intact ␣V8-10 was known to elute (22). Sequence analysis confirmed the presence of ␣V8-10 in these fractions. The presence of 3 H in the flow-through indicated that most of the 3-[ 3 H]azioctanol incorporated into ␣V8-10 was not stably incorporated under the conditions of HPLC. To localize the 3 H incorporation within ␣V8-10 that was stably incorporated, 3-[ 3 H]azioctanol labeled ␣V8-10 that had been eluted from gel was digested with trypsin, under conditions known to cleave the fragment at ␣Lys-400 (22). HPLC purification of the digest showed the major peak of 3 H in the flow-through, as well as a peak of 3 H at fractions 30 -33 (Fig.  9A). Based upon the 3 H elution profile seen when intact ␣V8-10 was purified by HPLC, the 3 H in the flow-through, ϳ60% of the eluted 3 H, was assumed to result from 3-[ 3 H]azioctanol incorporation, which was unstable to HPLC conditions. The 3 H present between fractions 30 -33 accounted for ϳ15% of the total eluted 3 H. Sequence analysis of the pooled fractions 30 -33 showed the presence of a primary sequence beginning at ␣Tyr-401 (Ϫcarb: I 0 ϭ 502 pmol; ϩ␣BgTx: I 0 ϭ 457 pmol; ϩcarb: I 0 ϭ 423 pmol), near the beginning of ␣M4, along with a secondary sequence beginning at ␣Ser-388 (Ϫcarb: I 0 ϭ 68 pmol; ϩ␣BgTx: I 0 ϭ 70 pmol; ϩcarb: I 0 ϭ 72 pmol) (Fig. 9B). In all conditions tested, 3 H release was observed in cycles 8 and 12, indicating incorporation in ␣His-408 and ␣Cys-412. Additional low level release was seen reproducibly in cycle 3, corresponding to ␣Ala-403. been another residue or residues in ␣V8-10 that were labeled more prominently, but the incorporation at this site(s) was highly labile under the conditions of HPLC. DISCUSSION

3-[ 3 H]Azioctanol photoincorporates with high efficiency into
the ␣-subunit of the nAChR, with the primary site of incorporation being ␣Glu-262, within the ion channel at the extracellular end of ␣M2. Additional incorporation was present in ␣His-408 and ␣Cys-412, residues previously identified as being situated at the lipid protein interface (19,24), and in ␣Tyr-190 and ␣Tyr-198, residues at the agonist binding site (2), as well as minor incorporation elsewhere. Whereas the incorporation in ␣M4 was independent of the presence of other drugs, the incorporation at ␣Glu-262 increased for nAChR in the desensitized state, and incorporation at ␣Tyr-190/␣Tyr-198 was seen only in the absence of carbamylcholine or ␣BgTx.
When labeling was analyzed at the level of the subunit, the most prominent pharmacology of labeling was the dependence of the ␣-subunit incorporation on the presence of carbamylcholine. This increased incorporation was because of the desensitization of the nAChR because other agonists also increased the incorporation, whereas the incorporation was lowest in the presence of pancuronium or ␣BgTx. The competitive antagonists d-tubocurarine and gallamine caused only a partial increase in the incorporation in the ␣-subunit. In the presence of carbamylcholine, the aromatic amine noncompetitive antagonist meproadifen partially (ϳ60%) inhibited the incorporation in the ␣-subunit, although two other aromatic amine noncompetitive antagonists, phencyclidine and QX-222, did not.

3-[ 3 H]
Azioctanol incorporated into the ␣-subunit at several sites. The levels of incorporation at these sites are summarized in Table I. The incorporation was calculated from mass levels and radioactivity incorporation from duplicate labeling experiments, although the values were determined differently for the large subunit fragments produced by V8 protease, their subfragments, and individual labeled amino acids. To estimate incorporation by sequence analysis in large subunit fragments (␣V8-20, ␣V8-18, and ␣V8-10) or in fragments isolated by HPLC, incorporation was calculated as the 3 H loaded on the sequencer filter divided by three times the observed initial yield of the sequence. This calculation is likely an overestimate because it is unknown what percent of the loaded material was sequencable, perhaps as little as 10% or maybe up to about 50%. An additional source of error for the calculation of incorporation in the ␣V8-20 and ␣V8-10 fragments was the necessity of treating the samples on the filter to remove excess SDS before sequencing. For incorporation at individual amino acids, the mass of that residue was calculated from the initial and repetitive yields. In this calculation, the radioactivity released and the mass levels reflect only the sequenced material. Because of the differences in the calculations, comparisons should only be made between values determined by the same method. For example, the incorporation in the subfragment containing ␣M2 from ␣Met-243 to ␣Lys-340, was calculated to be ϳ1.4 mol/mol whereas the incorporation at ␣Glu-262 was only ϳ0.35 mol/mol. However, the lack of significant incorporation in any other amino acids in this fragment indicate that it is unlikely that the incorporation at ␣Glu-262 accounts for only one-third of the incorporation in the fragment. Additionally, the incorporation in ␣V8-10 is much greater than the incorporation in the isolated fragments. As is discussed later, this discrepancy is because of lack of stability of incorporation to HPLC conditions. The primary site of incorporation of 3-[ 3 H]azioctanol was ␣Glu-262. This residue was the only residue labeled whose incorporation at 1 and 275 M 3-[ 3 H]azioctanol (Table I) did not increase approximately linearly with concentration. In the de-sensitized nAChR, ␣Glu-262 was labeled at ϳ6% at 1 M and ϳ33% at 275 M, an increase of only ϳ6-fold. The other sites, those in ␣M4 and the agonist site, showed an ϳ100-fold increase between 1 and 275 M, indicating a lack of saturation at these sites at these concentrations. The incorporation at multiple sites of varying affinities could account for differences between the half-maximum calculated by the incorporation in ␣-subunit, ϳ0.8 mM, and that calculated from flux assays (K ϳ100 M). 2 This discrepancy might alternatively result from differences in the experimental conditions, because the labeling experiments were carried out at higher protein concentrations (2 mg/ml) than the flux assays (50 ng/ml). However, the incorporation in ␣Glu-262 does saturate within the concentration range expected for the inhibition of the nAChR.
The specific radioactivity incorporation of most noncompetitive antagonist affinity reagents into the channel lumen can be inhibited by the presence of high concentrations of the nonradioactive analog. However, 1 mM octanol failed to inhibit the incorporation of 3-[ 3 H]azioctanol. The lack of inhibition could be because of separate binding sites for octanol and 3-azioctanol, although a binding to a common site is also consistent with the data. If octanol behaves similarly to 3-azioctanol, little inhibition is expected at 1 mM octanol, near the concentration at which the incorporation of 3-[ 3 H]azioctanol in the ␣-subunit was half-maximal, ϳ0.8 mM. Limits on the solubility of octanol prevented experiments at higher concentrations. The rapid binding and unbinding of octanol during photolysis could also contribute to the lack of inhibition by octanol.
Like 3-[ 3 H]azioctanol, [ 3 H]meproadifen mustard incorporated into ␣Glu-262 (16). The incorporation was fully inhibited by other charged noncompetitive antagonists, indicating that these drugs bind in a mutually exclusive manner, perhaps because of charge-charge repulsion. However, under conditions where meproadifen should fully occupy its site in the channel domain, it only reduced incorporation of 3-[ 3 H]azioctanol into ␣Glu-262 by ϳ40%. Further experiments will be needed to determine whether these two ligands can bind simultaneously.
Whereas the primary pharmacology observed at the level of the intact subunit was the increased incorporation in the presence of carbamylcholine attributable to increased incorporation at ␣Glu-262, analysis of subunit fragments revealed that there was also photolabeling of ␣Tyr-190 and ␣Tyr-198 inhibitable by the presence of carbamylcholine or ␣BgTx. These residues have both been labeled previously by competitive antagonists, such as d- [ (28). Because 3-[ 3 H]azioctanol labeled the agonist site, but only in the absence of carbamylcholine or ␣BgTx, it is likely that the inhibition of agonist binding by alcohols is because of direct low affinity competition of the alcohol with the agonist for the agonist binding site.
Although the photolabeling of sites outside the M2 segment showed no evidence of saturating incorporation between 1 and 275 M 3-[ 3 H]azioctanol, it is possible that the absolute levels of incorporation in a given residue were underestimated. Whereas the incorporation in ␣V8-20 and ␣V8-18 appeared stable under the HPLC conditions used, ϳ60% of the 3 H in the ␣V8-10 fragment was eluted in the flow-though of the HPLC. Therefore, at all concentrations the incorporation at ␣His-408, ␣Cys-412, or ␣Ala-403 or possibly another site, was most likely underestimated, because of instability of the photoadduct.
The high efficiency of the incorporation of 3-[ 3 H]azioctanol into ␣Glu-262 could be because of preferential reactivity of 3-[ 3 H]azioctanol with glutamates. However, whereas the only other reported amino acid labeled by an aliphatic diazirine was a glutamate of a hexosaminidase (29), in our study 3-[ 3 H]azioctanol photoincorporated into a variety of side chains, including histidine, cysteine, alanine, valine, and tyrosine. Acidic side chains near labeled side chains in other fragments showed no 3-[ 3 H]azioctanol incorporation. For example, in ␣M4 there was reactivity with alanine, cysteine, and histidine, but no reaction with ␣Asp-407, adjacent to the labeled histidine at the N terminus of ␣M4. Therefore, the high reactivity with ␣Glu-262 is most likely due primarily to a higher affinity of 3-[ 3 H]azioctanol for that region of the ion channel.
The preferential labeling of ␣M2 by 3-[ 3 H]azioctanol contrasts with the labeling of homologous residues in multiple subunits seen for many other noncompetitive antagonists (including [ 3 H]chlorpromazine, [ 3 H]triphenylmethylphosphonium, 3-(trifluoro-methyl)-3-(m-[ 125 I]iodophenyl)diazirine, and [ 3 H]tetracaine (2, 30)) which labeled amino acids in the M2 segment of each subunit. However, meproadifen mustard also reacted selectively with ␣Glu-262 in the desensitized state. Although the observed preference may be partially attributable to higher reactivity of the reactive intermediate with glutamates, in the ␤-subunit the equivalent residue is an aspartate, which should not react very differently from a glutamate. This position in ␤, however, when mutated to a cysteine, is not modified by water-soluble modification reagents whereas a cysteine at ␣-262 is (31,32). Therefore, the three-dimensional structure of the nAChR ␤-subunit, and perhaps the ␥and ␦-subunits, is not similar to that of ␣ in this region of the ion channel domain, and the preferential incorporation into the ␣-subunit may reflect a unique conformation of the ␣-subunit in this region.
Mutational analyses have identified amino acids in the nAChR as well as different positions within the inhibitory GABA A and glycine receptors which contribute to anesthetic potency. Previous studies aimed at elucidating the site of long chain alcohol binding on the nAChR have implicated residues at the 10Ј-position within the lumen of the channel (33). Within the GABA A and glycine receptors, which, in general, are potentiated by general anesthetics, two residues, one in the M2 segment, the other in M3, are known to confer sensitivity to several classes of anesthetics, including long chain alcohols, volatile anesthetics (isoflurane and enflurane), and intravenous anesthetics (etomidate) (reviewed in Ref. 34). The position in M2, 15Ј (see Fig. 10), is located in the extracellular half of the M2 segment on the face of the M2 ␣-helix opposite the lumen of the ion channel. The position in M3 is about seven amino acids from the N terminus of M3, but the orientation of this segment is not clearly established. However, this residue has been predicted to face the M2 helix, positioning it near the M2 15Ј residue (5). Neither of these residues was labeled in the nAChR by 3-[ 3 H]azioctanol. The lack of labeling would be consistent with different binding sites on the two receptors, reflecting the different actions of long chain alcohols on them. Fig. 10 shows a model of the nAChR ␣M2 segment as an ␣-helix including the positions implicated by mutational work on the nAChR and GABA A receptor and the photolabeled residue reported here. The azi group of 3-azioctanol was positioned near ␣Glu-262 (20Ј). The carbon chain of 3-azioctanol reaches to the 13Ј residues but does not reach the 10Ј position which is a determinant of alcohol potency in the nAChR. For the nAChR then, the photolabeling and mutagenesis results are in apparent disagreement. However, the nAChR mutagenesis studies measure octanol inhibition of the open state of the receptor, whereas the photoaffinity labeling studies are done with a desensitized receptor. It is possible that octanol binds at different positions in the two states. Indeed, differential labeling of the M2 ion channel in the resting and desensitized states has been seen with two other photoaffinity probes, 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine (35) and [ 3 H]diazofluorene (24). 3-Azioctanol might bind closer to the 10Ј-position in the open state, and closer to ␣Glu-262 in the desensitized state. Alternatively, the mutations studied may have changed the structure of the region near ␣Glu-262.
The studies presented here provide strong evidence that, in the desensitized state of the nAChR, the highest affinity binding site of 3-[ 3 H]azioctanol is within the ion channel domain near ␣Glu-262. Further studies, such as photoincorporation of 3-[ 3 H]azioctanol in the open channel and the effects of sitedirected mutagenesis of the N-terminal end of the M2 segment on the inhibition of the nAChR by alcohols, will be necessary to further define the site of action of long chain alcohols on the nAChR.