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J. Biol. Chem., Vol. 275, Issue 38, 29441-29451, September 22, 2000

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

Megan B. Pratt, S. Shaukat Husain^§, Keith W. Miller^§, and Jonathan B. Cohen^¶

From the  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

Received for publication, May 31, 2000, and in revised form, June 16, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 -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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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_A¹ and glycine receptors. They also noncompetitively inhibit excitatory receptors such as nicotinic acetylcholine receptors (nAChR) and serotonin 5HT₃ 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 (2-4).

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 (5-7). 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 10' (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 EC₅₀ of ~160 µM, an EC₅₀ 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 µM 3-[³H]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-[³H]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 µM 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- nAChR-enriched membranes were isolated from Torpedo californica electric organ (16). The final membrane suspensions were stored in 38% sucrose at 80 °C under argon. The membranes used here contained 0.5-2.0 nmol of acetylcholine binding sites per milligram of protein. 3-[³H]Azioctanol and nonradioactive 3-azioctanol were synthesized as described previously (15). The specific activity of the 3-[³H]azioctanol was ~11 Ci/mmol. This stock was stored at 20 °C in CH₂Cl₂, which was removed via evaporation immediately prior to the addition of membranes or isotopic dilution. For studies of incorporation at concentrations higher than 1 µM 3-[³H]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 CaCl₂, 2 mM MgCl₂, 5 mM sodium phosphate, pH 7.0), to a final specific activity of ~0.04 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-[³H]Azioctanol-- For analytical labeling experiments, freshly thawed Torpedo membranes were diluted with TPS and pelleted (15000 × g) for 30 min, and the pellets were resuspended in TPS at 2 mg/ml protein (~1 µM nAChR). Membrane aliquots (100 µg per condition) were combined with 3-[³H]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-[³H]azioctanol-labeled -subunit with S. aureus V8 protease (17, 18), labeling was carried out with 400 µg (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 Me₂SO) was added to a final concentration of 500 µM. 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 × 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 80 °C to Eastman Kodak X-OMAT film for various times (6-8 weeks). Additionally, incorporation of ³H into individual polypeptides was quantified by scintillation counting of excised gel slices (21). For analytical V8-mapping 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 ³H 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 ~20 kDa (V8-20) and ~10 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 NH₄HCO₃, 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 20 °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 µl. 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 µl) of 100 mM NH₄HCO₃, 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-[³H]azioctanol were further purified by reverse-phase HPLC (22), using a Brownlee C4-Aquapore column (100 × 2.1 mm, 7-µm 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 ³H by liquid scintillation counting.

Intact V8-18 and V8-18 proteolytic fragments were also purified by HPLC (23), using a Brownlee C4-Aquapore column. Solvent A was 0.09% trifluoroacetic acid in water, and solvent B was 0.1% trifluoroacetic acid in acetonitrile. A linear gradient with several steps was used: 0 min, 10% solvent B; 10 min, 10% solvent B; 25 min, 25% solvent B; 45 min, 40% solvent B; 65 min, 60% solvent B; 75 min, 100% solvent B. The rate of flow was 0.25 ml/min, and 0.5-ml fractions were collected. Measurements were determined as for the purification of the fragments of enzymatic digestion.

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-µl fractions) were directly loaded onto chemically modified glass fiber disks (Beckman) in 20-µl 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 = I₀ × Rⁿ, where M is the observed release, I₀ 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 ³H 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 ³H 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 ³H than for mass). In this calculation, the radioactivity released and the mass levels reflect only the sequenced material.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Photoincorporation of 3-[³H]Azioctanol into nAChR-rich Membranes-- Initial experiments were designed to characterize the general pattern of photoincorporation of 3-[³H]azioctanol and to test the sensitivity of photoincorporation to various ligands. For these initial experiments, two concentrations of 3-[³H]azioctanol were used, 1 µM (11 Ci/mmol) and 275 µM (0.04 Ci/mmol). For inhibition of Torpedo nAChR using flux assays, the IC₅₀ of 3-azioctanol is ~100 µM.² Therefore, 1 µM 3-[³H]azioctanol was well below the concentration necessary for inhibition of 50% of the nAChR, whereas 275 µM 3-[³H]azioctanol was a concentration sufficient to produce greater than 50% inhibition. Isotopic dilution of 3-[³H]azioctanol resulted in the presence of similar levels of ³H in the samples containing 1 µM and 275 µM 3-[³H]azioctanol. Membranes (2 mg/ml protein) were equilibrated with 3-[³H]azioctanol in the presence and absence of 2 mM carbamylcholine. After irradiation for 10 min at 365 nm, the pattern of incorporation was assessed by SDS-PAGE followed by fluorography or scintillation counting of gel slices.

As seen in the fluorograph of the 8% polyacrylamide gel (Fig. 1A) at both 3-[³H]azioctanol concentrations in the absence of carbamylcholine, the principal polypeptide labeled was a 34-kDa polypeptide identified as a mitochondrial chloride channel (VDAC) (24). The ³H incorporation in VDAC, although not affected by the presence of carbamylcholine, was reduced by 50% at the higher concentration of 3-[³H]azioctanol. This decrease suggests specific incorporation of 3-[³H]azioctanol in VDAC, which is inhibited by excess non-radioactive 3-azioctanol. The ³H incorporation in other non-receptor polypeptides (rapsyn (43 kDa) and the -subunit of (Na⁺/K⁺)-ATPase (NK)) was not altered by the presence of carbamylcholine and appeared similar at 1 and 275 µM 3-[³H]azioctanol.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Photoincorporation of 3-[3H]azioctanol into nAChR-rich membranes in the presence or absence of carbamylcholine. A, nAChR-rich membranes (100 µg at 2 mg/ml) were equilibrated with 1 µM (11 Ci/mmol) (lanes 2 and 3) and 275 µM (0.04 Ci/mmol) (lanes 4 and 5) 3-[3H]azioctanol in TPS, in the absence (lanes 2 and 4) or presence (lanes 3 and 5) of 2 mM carbamylcholine and irradiated at 365 nm for 10 min. After photolysis, samples were subjected to SDS-PAGE, visualized by Coomassie Blue (lane 1), processed for fluorography, and exposed to film for 6 weeks (lanes 2-5). Indicated on the left are the mobilities of nAChR subunits, rapsyn (43 kDa), the -subunit of the (Na+/K+)-ATPase (NK), and the mitochondrial chloride channel (34 kDa). B, 3H incorporated in the absence or presence of carbamylcholine (carb) at 1 and 275 µM 3-[3H]azioctanol was quantified by scintillation counting as described under "Experimental Procedures." Bars shown are the mean ± S.D. of duplicate samples.

Of the nAChR subunits,  was labeled most strongly. Incorporation of 3-[³H]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 ~5-fold at 1 µM 3-[³H]azioctanol (Fig. 1B) and ~3-fold at 275 µM. The presence of agonist also increased the incorporation in the -subunit, although only by ~1.4-fold at 1 µM. Because the 3-[³H]azioctanol at 275 µM had an ~275-fold lower specific activity, the observed similarity in the ³H incorporation in the -subunit at the two conditions indicated that the -subunit labeled in the presence of 275 µM 3-[³H]azioctanol contained ~275-fold more moles of 3-azioctanol per mol of subunit.

The enhancement of 3-[³H]azioctanol photolabeling in the -subunit by carbamylcholine as well as other cholinergic agonists and competitive antagonists (Fig. 2) was determined by quantification of ³H incorporation in gel slices. At concentrations sufficient to fully occupy the ACh site, the agonists phenyltrimethylammonium and nicotine increased 3-[³H]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 ~60% of that seen in the presence of carbamylcholine, whereas for pancuronium, a competitive antagonist that is not known to desensitize the receptor, 3-[³H]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-[³H]azioctanol into non-nAChR polypeptides including rapsyn (43 kDa), calelectrin (37 kDa), or the (Na⁺/K⁺)-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 = 4 µM (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 µM (27), this discrepancy was not unexpected because the photolabeling experiment was carried out at a concentration of ACh sites of 2.2 µM.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effects of nAChR agonists and competitive antagonists on the photoincorporation of 3-[3H]azioctanol into nAChR-rich membranes. nAChR-rich membranes (100 µg at 2 mg/ml) were equilibrated with 1 µM 3-[3H]azioctanol in TPS in the absence of other drugs or in the presence of 2 mM carbamylcholine (carb), 200 µM phenyltrimethylammonium (PTA), 100 µM nicotine, 100 µM pancuronium, 1 mM gallamine, or 30 µM d-tubocurarine and irradiated for 10 min at 365 nm. After photolysis, samples were subjected to SDS-PAGE and visualized by Coomassie Blue. Bands corresponding to the -subunit as well as the 37-kDa (calelectrin) and 43-kDa (rapsyn) bands were excised. 3H incorporation was quantified by scintillation counting. Bars indicate the mean ± S.D.

The effects of several noncompetitive antagonists on the incorporation of 3-[³H]azioctanol at 1 µM were also tested (Fig. 3A). For membranes equilibrated with carbamylcholine, the ³H incorporation in the nAChR -subunit was insensitive to the presence of 1 mM octanol. At 100 µM, meproadifen, an aromatic amine noncompetitive antagonist, reduced the incorporation by ~50%. Two other aromatic amine noncompetitive antagonists, phencyclidine and QX-222, failed to inhibit the incorporation of 3-[³H]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).

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Effects of nAChR noncompetitive antagonists on the photoincorporation of 3-[3H]azioctanol into nAChR-rich membranes. nAChR-rich membranes (100 µg at 2 mg/ml) were labeled with 1 µM (11 Ci/mmol) or 275 µM (0.04 Ci/mmol) 3-[3H]azioctanol. A, at 1 µM 3-[3H]azioctanol, membranes were labeled in the absence of other drugs, in the presence of 2 mM carbamylcholine (carb) with no other drug, or with 1 mM octanol or 100 µM meproadifen. B, at 275 µM 3-[3H]azioctanol, membranes were labeled in the absence or the presence of 2 mM carbamylcholine or 10 µM BgTx in the absence or presence of 100 µM meproadifen. Following irradiation at 365 nm for 10 min, samples were subjected to SDS-PAGE and visualized by Coomassie Blue. Bands corresponding to nAChR -subunit and the 43-kDa (rapsyn) polypeptide were excised. 3H was quantified by scintillation counting.

The effects of meproadifen were also studied in the presence of 275 µM 3-[³H]azioctanol (Fig. 3B). At that concentration the presence of carbamylcholine resulted in an ~3-fold increase in the incorporation of 3-[³H]azioctanol in the -subunit over that seen in the absence of carbamylcholine. In the presence of carbamylcholine, meproadifen reduced the incorporation by ~50%. In the absence of carbamylcholine, meproadifen actually enhanced the 3-[³H]azioctanol incorporation. In the presence of BgTx, meproadifen did not alter the 3-[³H]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-[³H]azioctanol in nAChR -subunit was measured over a range of 3-[³H]azioctanol concentrations, using a constant specific activity of 3-[³H]azioctanol (Fig. 4). The incorporation in the (Na⁺/K⁺)-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 ~1 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 ~2 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 higher incorporation in the absence of carbamylcholine showed high variability. The total ³H incorporation at ~2 mM 3-[³H]azioctanol was ~0.25 mol of 3-[³H]azioctanol/mol of , based on the reported counting efficiency (25%) of the toluene-based gel mixture used (21).

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Concentration dependence of 3-[3H]azioctanol photoincorporation into nAChR -subunit. nAChR-rich membranes (100 µg at 2 mg/ml) were equilibrated with varying concentrations of 3-[3H]azioctanol (~0.04 Ci/mmol) in the absence of other drugs (, ), in the presence of 2 mM carbamylcholine (, ), or in the presence of 10 µM BgTx (, ). After irradiation at 365 nm for 10 min, samples were subjected to SDS-PAGE and visualized by Coomassie Blue. Bands corresponding to nAChR -subunit (solid symbols), as well as the 90-kDa band containing the -subunit of (Na+/K+)-ATPase (open symbols) were excised, and 3H incorporation was quantified by scintillation counting. Error bars are from the average of four separate experiments normalized to a common specific activity by assuming common level of incorporation in -subunit in the presence of carbamylcholine at 2.2 mM 3-[3H]azioctanol.

Mapping of 3-[³H]Azioctanol Photoincorporation into -Subunit Proteolytic Fragments-- The distribution of 3-[³H]azioctanol incorporation within the -subunit was examined by digestion of the labeled subunit with S. aureus V8 protease under conditions that are known to generate four large non-overlapping fragments resolvable by SDS-PAGE (Fig. 5, inset). The largest fragment, a 20-kDa peptide (V8-20), begins at Ser-173 and contains the first three membrane spanning regions, M1, M2, and M3 (18). The 10-kDa peptide (V8-10) contains the fourth membrane spanning region, M4, and begins at Asn-339. The 18-kDa (V8-18) and 4-kDa (V8-4) peptides begin at Val-46 and Ser-1, respectively. Membranes labeled with 3-[³H]azioctanol were subjected to SDS-PAGE, and the -subunit was excised. This gel piece was loaded onto a mapping gel along with V8 protease. The -subunit was cleaved in the gel with the protease, and the fragments were separated on the gel. Again, like the studies of the incorporation in the intact subunits, the incorporation was measured at both 1 µM and 275 µM 3-[³H]azioctanol. Similar levels of ³H were used at the two concentrations, with a specific activity of 11 Ci/mmol at 1 µM and 0.04 Ci/mmol at 275 µM, an ~275-fold reduction in specific activity in the samples labeled in the presence of 275 µM 3-[³H]azioctanol compared with those labeled under the 1 µM condition. Based on liquid scintillation counting of these -subunit proteolytic fragments, the main sites of photoincorporation in the absence of agonist were within the V8-20³ and V8-10 fragments (Fig. 5). The ³H incorporation in each fragment was similar at both concentrations of 3-[³H]azioctanol. In the absence of agonist, the incorporation in V8-10 was ~60% that of V8-20. The addition of agonist increased the labeling of the V8-20 fragment, 9-fold at 1 µM and 5-fold at 275 µM, whereas the ³H incorporated in V8-10 was unchanged by the presence of carbamylcholine. In the presence of carbamylcholine, the incorporation in V8-20 accounted for ~90% of the incorporation at both 3-[³H]azioctanol concentrations, whereas V8-10 contained ~6% of the total 3-[³H]azioctanol incorporation within the -subunit fragments. The similar levels of ³H incorporation in the fragments between the two concentrations, with 3-[³H]azioctanol at an ~275-fold lower specific activity at 275 µM, indicated that ~275-fold more molecules of 3-azioctanol were incorporated at 275 µM 3-[³H]azioctanol. In all conditions, the incorporation in V8-18 and V8-4 appeared similar and was lower than the incorporation in V8-10.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Mapping of sites of 3-[3H]azioctanol incorporation into the nAChR -subunit using S. aureus V8 protease. nAChR-rich membranes (400 µg at 2 mg/ml) were labeled with 1 µM (11 Ci/mmol) or 275 µM (0.04 Ci/mmol) 3-[3H]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 3H incorporation was quantified by scintillation counting. 3H present in proteolytic fragments of nAChR -subunit labeled in the absence (solid and vertical hatched bars) or presence of 2 mM carbamylcholine at 1 µM (solid and open bars) and 275 µM (vertical and horizontal hatched bars) 3-[3H]azioctanol is shown. Inset, the nAChR -subunit proteolytic fragments produced by digestion by V8 protease.

The carbamylcholine-dependent labeling of nAChR with 3-[³H]azioctanol was in the V8-20 fragment containing M1, M2, and M3. To further localize the site of labeling, 10 mg of membranes were labeled with 1 µM or 275 µM 3-[³H]azioctanol in the presence or absence of carbamylcholine, meproadifen, or BgTx. Additionally, these membranes were labeled with 1-azidopyrene, a fluorescent compound that photoincorporates in transmembrane segments, to aid in the localization of transmembrane segments. Following the digestion of -subunit with V8 protease, the V8-20, V8-18, and V8-10 fragments were excised and eluted. To quantify the ³H incorporation, the eluted V8-20 and V8-10 fragments were subjected to sequence analysis. Based on sequence analysis of the fragments, at 1 µM 3-[³H]azioctanol, in the absence of carbamylcholine ~0.008 moles of 3-[³H]azioctanol incorporated into a mole of V8-20 and ~0.004 moles into V8-10. In the presence of carbamylcholine, 0.06 moles incorporated into V8-20 and 0.004 moles into V8-10. At 275 µM 3-[³H]azioctanol, the incorporation increased with ~0.55 moles incorporated per mole of V8-20 and 0.24 moles per mole V8-10 in the absence of carbamylcholine. In the presence of carbamylcholine at 275 µM 3-[³H]azioctanol, ~1.3 moles 3-[³H]azioctanol incorporated into V8-20 and ~0.40 moles into V8-10.

3-[³H]Azioctanol Photoincorporation within the M2 Segment-- To determine whether there was incorporation in the M2 segment, the eluted V8-20 fragment, labeled with 3-[³H]azioctanol, was digested with EndoLysC. Digestion with EndoLysC is known to create an ~10-kDa fragment starting at Met-243, the N terminus of the M2 segment, that can be purified by reverse-phase HPLC (16). When the EndoLysC-digested V8-20, which had been labeled with 275 µM 3-[³H]azioctanol in the presence of carbamylcholine, was fractionated by reverse-phase HPLC, ~80% of the ³H eluted in a peak centered at fraction 33 (~88% organic) (Fig. 6A). For the samples labeled in the presence of BgTx or the absence of other drugs, the ³H in fraction 33 was only ~20% that seen for the sample labeled in the presence of carbamylcholine.

View larger version (33K): [in this window] [in a new window]   Fig. 6.   HPLC purification and sequence analysis of 3-[3H]azioctanol-labeled fragments from an EndoLysC digest of V8-20. A, V8-20 isolated from nAChRs photolabeled with 275 µM 3-[3H]azioctanol in the absence () or presence of 10 µM BgTx () or 2 mM carbamylcholine () was digested with EndoLysC. The digest was applied to a Brownlee Aquapore-C4 column and fractionated by reverse-phase HPLC. Upper, 3H elution profiles (5% of each fraction counted). Lower, fluorescence (···) and absorbance () profiles. B and C, 3H (, , ) and mass released (, ) on N-terminal sequencing of material in HPLC fraction 33 (B) and 29 (C). B, fraction 33 from the samples labeled in the absence (, ) and presence of carbamylcholine (, ) showed a single sequence, beginning at Met-243, the N terminus of the M2 segment (carb: I0 = 23 pmol, R = 92%, 9800 cpm loaded and 3900 cpm remaining after 30 cycles; +carb: I0 = 30 pmol, R = 92%, 26000 cpm loaded and 3900 cpm remaining after 30 cycles). C, fraction 29 from the sample labeled in the absence (, ) or presence of BgTx () or carbamylcholine (, ) showed a primary sequence beginning at His-186 and a secondary sequence beginning at Asp-180 (carb: His-186 I0 = 35 pmol, R = 93%, Asp-180 I0 = 4.6 pmol, R = 86%, 16700 cpm loaded and 3400 cpm remaining after 25 cycles; +BgTx: His-186 I0 = 55 pmol, R = 93%, Asp-180 I0 = 2.4 pmol, R = 95%, 4100 cpm loaded and 1000 cpm remaining after 25 cycles; +carb: His-186 I0 = 36 pmol, R = 95%, Asp-180 I0 = 7.8 pmol, R = 82%, 4800 cpm loaded and 1200 cpm remaining after 25 cycles). Mass levels of released PTH-amino acids for BgTx not shown. Primary sequence for each fraction is shown on top axes.

For each labeling condition, fraction 33, which contained the peak of ³H 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 (carb: I₀ = 23 pmol; +carb: I₀ = 30 pmol). No other sequences were present at more than 10% the mass of the peptide beginning at Met-243. For the sample labeled in the presence of carbamylcholine, there was a peak of ³H release in cycle 20, corresponding to incorporation at Glu-262, and that release was reduced by ~60% in the sample labeled in the absence of carbamylcholine or in the presence of BgTx (data not shown). Based upon the ³H release in cycle 20, in the presence of carbamylcholine, there was ~0.33 mol of 3-[³H]azioctanol incorporated per mol of Glu-262. In the absence of other drugs or in the presence of BgTx, there was ~0.14 mol of 3-[³H]azioctanol incorporated per mol of Glu-262.

The HPLC profile of the EndoLysC-digest of V8-20 labeled in the presence of 1 µM 3-[³H]azioctanol was similar to that at 275 µM (data not shown). For the sample labeled in the presence of carbamylcholine, ~70% of the ³H eluted as a single peak at ~90% organic. As with fraction 33 from the sample labeled in the presence of 275 µM 3-[³H]azioctanol, sequence analysis of the fraction containing the peak of ³H from the samples labeled with 1 µM 3-[³H]azioctanol revealed the presence of a single sequence beginning at Met-243 with release of ³H in cycle 20 (data not shown). In the presence of carbamylcholine, the release in cycle 20 was equivalent to 0.025 mol per mol of Glu-262 and that labeling was reduced by ~40% for the sample labeled in the presence of meproadifen and carbamylcholine (0.014 mol of 3-[³H]azioctanol per mol of Glu-262). In the absence of carbamylcholine, the incorporation of 3-[³H]azioctanol at Glu-262 (0.0012 mol of 3-[³H]azioctanol incorporated per mol of Glu-262) was ~5% that seen in the presence of carbamylcholine.

3-[³H]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 µM 3-[³H]azioctanol in the presence of carbamylcholine indicated the presence of the sequence beginning at His-186 (I_o = 36 pmol) (see Fig. 6C), containing ~0.05 mol of incorporated label per mol of fragment. This incorporation is ~4% 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.

Solution digestion of V8-20 with V8 protease generates an ~9-kDa fragment that begins at Leu-263 (the N terminus of the M2-M3 linker) and contains the M3 segment (19). To determine whether 3-[³H]azioctanol incorporated into the M3 segment, V8-20 labeled with 275 µM 3-[³H]azioctanol was digested with V8 protease, and the fragments were separated by HPLC (Fig. 7). V8 protease cleaves at the C-terminal side of glutamate and to generate the fragment beginning at Leu-263, cleavage must occur at Glu-262, which is labeled by 3-[³H]azioctanol. Therefore, it was expected that only fragments not labeled at Glu-262 would be digested to generate the fragment beginning at Leu-263. In the sample labeled in the presence of carbamylcholine, ~85% of the ³H eluted at fraction 33 (Fig. 7, inset). This fraction, based on sequence analysis, contained a fragment beginning at the N terminus of V8-20, and based on the high levels of ³H in the fraction, this fragment should have contained the M2 segment. The fragment beginning at Leu-263 was expected to elute at ~55% organic (19). A small peak of ³H was present in fraction 23 (~50% organic), and one-half of this fraction from each condition was subjected to Edman degradation (data not shown). Two sequences were present, the first fragment beginning at Leu-263 (carb: I₀ = 4.8 pmol; +BgTx: I₀ = 3.4 pmol; +carb: I₀ = 1.5 pmol) and a fragment beginning at Thr-52 (carb: I₀ = 72 pmol; +BgTx: I₀ = 24 pmol; +carb: I₀ = 37 pmol), an N terminus of the V8-18 fragment arising from contamination of the V8-20 sample with V8-18. Based upon the mass levels present, if the ³H in this fraction were attributable only to the sequence beginning at Leu-263, then, in the presence of carbamylcholine, ~0.08 mol of 3-[³H]azioctanol incorporated per mol of fragment, ~6% of the incorporation in the fragment beginning at Met-243. Therefore, the M3 segment was labeled at less than 6% the levels of incorporation in the M2 segment.

View larger version (35K): [in this window] [in a new window]   Fig. 7.   HPLC purification of 3-[3H]azioctanol-labeled fragments from S. aureus V8 protease digest of V8-20. V8-20 isolated from nAChRs labeled with 275 µM 3-[3H]azioctanol in the absence () or presence of 10 µM BgTx () or 2 mM carbamylcholine () was digested with V8 protease in solution, and the digest was fractionated by reverse-phase HPLC. Upper, 3H elution profiles (5% of each fraction). Inset, replot of 3H to include peak of 3H in fraction 33. Lower, fluorescence (···) and absorbance () profiles.

3-[³H]Azioctanol Photoincorporation within the Agonist Binding Site-- For the V8-20 fragment isolated from nAChRs labeled with either 1 or 275 µM 3-[³H]azioctanol in the absence of carbamylcholine, the HPLC chromatogram of the EndoLysC digest of V8-20 (Fig. 6A) contained a peak of ³H 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 (carb: I₀ = 35 pmol; +BgTx: I₀ = 55 pmol; +carb: I₀ = 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 µM 3-[³H]azioctanol, ³H 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 ³H 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 ~10% that in Glu-262 in the absence of carbamylcholine, with 3-[³H]azioctanol only incorporating at ~0.013 mol per mol of Tyr-190 and ~0.017 mol per mol of Tyr-198. A similar pattern of release, although with lower levels of ³H incorporation, was seen in the sample labeled with 1 µM 3-[³H]azioctanol in the absence of carbamylcholine (see 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 ~0.003 mol incorporated per mol of Val-189.

View this table: [in this window] [in a new window]   Table I Incorporation of 3-[3H]azioctanol into fragments and residues of the -subunit The ratio of moles 3-[3H]azioctanol incorporated per mol of the fragments or residues labeled was calculated from the 3H incorporation and the known specific activity of 3-[3H]azioctanol. The 3H 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.

3-[³H]Azioctanol Photoincorporation within V8-18-- To characterize the levels of incorporation with 275 µM [³H]azioctanol in the V8-18 fragment compared with the incorporation in V8-20, V8-18 was purified by reverse-phase HPLC (Fig. 8A). A peak of ³H eluted at fraction 23 as well as in two hydrophobic peaks. The hydrophobic peaks corresponded to contamination by V8-20. Sequence analysis of fraction 23 showed two sequences present at similar levels, one beginning at Val-46 (carb: I₀ = 41 pmol; +BgTx: I₀ = 19 pmol; +carb: I₀ = 32 pmol) and the other beginning at Thr-52 (carb: I₀ = 38 pmol; +BgTx: I₀ = 24 pmol; +carb: I₀ = 31 pmol) (Fig. 8B). These two peptides are the known N termini of V8-18 (18). Radioactive release was evident in the 6th cycle. Similar levels of release were seen in the presence or absence of carbamylcholine or BgTx. The residue, either Glu-51 or Arg-57, was labeled by ~0.003 mol of 3-[³H]azioctanol per mol of residue. Although both sequences were present at similar mass levels, the release is likely from labeling at Glu-51, the sixth cycle of the sequence beginning at Val-46. If the release were because of labeling of Arg-57, additional release would have been expected in cycle 12, corresponding to Arg-57 in the sequence beginning at Val-46.

View larger version (35K): [in this window] [in a new window]   Fig. 8.   HPLC purification and sequence analysis of 3-[3H]azioctanol-labeled V8-18. A, V8-18 isolated from nAChR labeled with 275 µM 3-[3H]azioctanol in the absence () or presence of 10 µM BgTx () or 2 mM carbamylcholine () was purified by reverse-phase HPLC. Upper, 3H elution profiles (5% of each fraction). Lower, fluorescence (···) and absorbance () profiles. B, 3H (, , ) and mass released (, ) on N-terminal sequencing of material from HPLC fraction 23. The sample labeled in the absence (, , ) or presence of BgTx () or carbamylcholine () showed two sequences, one beginning at Val-46 and one beginning at Thr-52 (carb: Val-46 () I0 = 41 pmol, R = 92%, Thr-52 () I0 = 38 pmol, R = 94%, 10120 cpm loaded and 2400 cpm remaining after 15 cycles; +BgTx: Val-46 I0 = 19 pmol, R = 91%, Thr-52 I0 = 24 pmol, R = 91%, 10320 cpm loaded and 2000 cpm remaining after 8 cycles; +carb: Val-46 I0 = 32 pmol, R = 92%, Thr-52 I0 = 31 pmol, R = 94%, 9800 cpm loaded and 1800 cpm remaining after 15 cycles). The two sequences that were present are shown along the top axis.

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 ³H that contained a single sequence, that beginning at Lys-77 (carb: I_o = 22 pmol, +BgTx I_o = 15 pmol, +carb: I_o = 13 pmol) (data not shown). The fragment showed ~10% incorporation in each of the three conditions. The incorporation in this fragment accounts for most of the incorporation in V8-18 because there was ~6% 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-[³H]azioctanol per mol of residue (data not shown).

3-[³H]Azioctanol Photoincorporation within V8-10-- At 275 µM 3-[³H]azioctanol, V8-10 fragments labeled in the presence or absence of other cholinergic drugs showed similar levels of ³H incorporation. Additionally, the levels of incorporation in V8-10 labeled with 1 µM 3-[³H]azioctanol were similar in the presence and absence of other drugs. HPLC purification of intact V8-10 labeled with 275 µM 3-[³H]azioctanol revealed that ~60% of the incorporated ³H eluted in the flow-through (Fig. 9A, inset), whereas only ~20% 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 ³H in the flow-through indicated that most of the 3-[³H]azioctanol incorporated into V8-10 was not stably incorporated under the conditions of HPLC.

View larger version (36K): [in this window] [in a new window]   Fig. 9.   HPLC purification and sequence analysis of 3-[3H]azioctanol-labeled fragments from trypsin digestion of V8-10. A, V8-10 labeled with 275 µM 3-[3H]azioctanol in the absence () or presence of 10 µM BgTx () or 2 mM carbamylcholine () was digested with trypsin, and the digest was fractionated by reverse-phase HPLC. Upper, 3H elution profiles (10% of each fraction). Lower, fluorescence (···) and absorbance () profiles. Inset, 3H elution profile of undigested V8-10 labeled with 1 µM 3-[3H]azioctanol in the presence of 2 mM carbamylcholine purified by reverse-phase HPLC. B, 3H (, , ) and mass released (, ) on N-terminal sequencing of material from HPLC fractions 31-34. The samples labeled in the absence (,  or presence of BgTx () or carbamylcholine (, ) showed a primary sequence beginning at Tyr-401 and a secondary sequence beginning at Ser-388 (carb: Tyr-401 I0 = 502 pmol, R = 90%, Ser-388 I0 = 68 pmol, R = 87%, 52400 cpm loaded and 12700 cpm remaining after 25 cycles; +BgTx: Tyr-401 I0 = 457 pmol, R = 89%, Ser-388 I0 = 70 pmol, R = 87%, 48500 cpm loaded and 16700 cpm remaining after 25 cycles; +carb: Tyr-401 I0 = 423 pmol, R = 90%, Ser-388 I0 = 72 pmol, R = 88%, 57800 cpm loaded and 19400 cpm remaining after 25 cycles). Mass levels of released PTH-amino acids for BgTx not shown. The primary sequence is shown along the top axis.

To localize the ³H incorporation within V8-10 that was stably incorporated, 3-[³H]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 ³H in the flow-through, as well as a peak of ³H at fractions 30-33 (Fig. 9A). Based upon the ³H elution profile seen when intact V8-10 was purified by HPLC, the ³H in the flow-through, ~60% of the eluted ³H, was assumed to result from 3-[³H]azioctanol incorporation, which was unstable to HPLC conditions. The ³H present between fractions 30-33 accounted for ~15% of the total eluted ³H. Sequence analysis of the pooled fractions 30-33 showed the presence of a primary sequence beginning at Tyr-401 (carb: I₀ = 502 pmol; +BgTx: I₀ = 457 pmol; +carb: I₀ = 423 pmol), near the beginning of M4, along with a secondary sequence beginning at Ser-388 (carb: I₀ = 68 pmol; +BgTx: I₀ = 70 pmol; +carb: I₀ = 72 pmol) (Fig. 9B). In all conditions tested, ³H 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. 3-[³H]Azioctanol incorporated into His-408 and Cys-412 at ~0.0025 mol per mol of residue at 275 µM and at ~1% that level at 1 µM. However, at both concentrations most of the ³H eluted with the flow-through of the HPLC, and this ³H could have been incorporated into these residues but labile under HPLC conditions. Alternatively, there could have 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

3-[³H]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 (~60%) inhibited the incorporation in the -subunit, although two other aromatic amine noncompetitive antagonists, phencyclidine and QX-222, did not.

3-[³H]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 ³H 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 ~1.4 mol/mol whereas the incorporation at Glu-262 was only ~0.35 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-[³H]azioctanol was Glu-262. This residue was the only residue labeled whose incorporation at 1 and 275 µM 3-[³H]azioctanol (Table I) did not increase approximately linearly with concentration. In the desensitized nAChR, Glu-262 was labeled at ~6% at 1 µM and ~33% at 275 µM, an increase of only ~6-fold. The other sites, those in M4 and the agonist site, showed an ~100-fold increase between 1 and 275 µM, 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, ~0.8 mM, and that calculated from flux assays (K ~100 µM).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-[³H]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-[³H]azioctanol in the -subunit was half-maximal, ~0.8 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-[³H]azioctanol, [³H]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-[³H]azioctanol into Glu-262 by ~40%. 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-[³H]tubocurarine and [³H]DDF, and agonists, such as [³H]nicotine (2). The efficiency of incorporation in these residues, 0.012% of Tyr-190 labeled at 1 µM and 1.3% labeled at 275 µM 3-[³H]azioctanol, was lower than the incorporation in Glu-262 even in the absence of carbamylcholine, 0.12% at 1 µM and 14% at 275 µM 3-[³H]azioctanol. The inhibition of this incorporation by the presence of carbamylcholine or BgTx established that the occupancy of the agonist binding site prevented the accessibility of 3-[³H]azioctanol to these side chains.

Between 1 and 275 µM 3-[³H]azioctanol, there was a linear increase in the level of incorporation at Tyr-190/Tyr-198 in the agonist site. Although no studies have been carried out with 3-[³H]azioctanol, octanol at concentrations up to 4 mM did not inhibit [³H]ACh binding, whereas butanol, which could be studied at high concentrations, inhibited the binding with a K_i of ~80 mM (28). Because 3-[³H]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 µM 3-[³H]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, ~60% of the ³H 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-[³H]azioctanol into Glu-262 could be because of preferential reactivity of 3-[³H]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-[³H]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-[³H]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-[³H]azioctanol for that region of the ion channel.

The preferential labeling of M2 by 3-[³H]azioctanol contrasts with the labeling of homologous residues in multiple subunits seen for many other noncompetitive antagonists (including [³H]chlorpromazine, [³H]triphenylmethylphosphonium, 3-(trifluoro-methyl)-3-(m-[¹²⁵I]iodophenyl)diazirine, and [³H]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 10'-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, 15' (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 15' residue (5). Neither of these residues was labeled in the nAChR by 3-[³H]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.

View larger version (37K): [in this window] [in a new window]   Fig. 10.   Model of 3-azioctanol and M2 helix. -Helical model of the M2 segment and space-filling model of octanol were made using the molecular modeling software Insight (Biosym, Inc.). M2 residues 10', 15', and 20' are shown as space-filling models. The diazirine of 3-azioctanol (dark) is positioned near Glu-262, the residue labeled by 3-[3H]azioctanol. The hydroxyl group is also shown darker than the other atoms.

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 (20'). The carbon chain of 3-azioctanol reaches to the 13' residues but does not reach the 10' 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-[¹²⁵I]iodophenyl)diazirine (35) and [³H]diazofluorene (24). 3-Azioctanol might bind closer to the 10'-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-[³H]azioctanol is within the ion channel domain near Glu-262. Further studies, such as photoincorporation of 3-[³H]azioctanol in the open channel and the effects of site-directed 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.

	ACKNOWLEDGEMENTS

The authors thank Aimée Powelka for contributions to the studies of the effects of agonists and competitive antagonists on 3-[³H]azioctanol incorporation in nAChR subunits.

	FOOTNOTES

^* 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.

^¶ 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.

Published, JBC Papers in Press, June 19, 2000, DOI 10.1074/jbc.M004710200

³ Staining of the mapping gel with Coomassie Blue revealed the four expected proteolytic fragments under all conditions. Additionally, a band was present at ~22 kDa under the condition labeled with 275 µM 3-[³H]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-[³H]azioctanol, resulting in ·reduced mobility. Therefore, the ³H present in this region was attributed to labeling in V8-20 and was added to that of the V8-20 band.

² M. A. Kloczewiak and K. W. Miller, unpublished data.

	ABBREVIATIONS

The abbreviations used are: GABA, -aminobutyric acid; 1-AP, 1-azidopyrene; nAChR, nicotinic acetylcholine receptor; PAGE, polyacrylamide gel electrophoresis; V8 protease, S. aureus glutamyl endopeptidase; EndoLysC, endoproteinase Lys-C; BgTx. -bungarotoxin, PTH, phenylthiohydantoin; VDAC, voltage dependent anion channel; carb, carbamylcholine; HPLC, high pressure liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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