Abstract
Noncompetitive antagonists (NCAs) 1 of the nicotinic acetylcholine receptor (AChR) are by definition agents that block the permeability response of the receptor by binding to sites that are distinct from the agonist-binding sites (recent reviews include Refs. 1-3). NCAs of the AChR are a large and structurally heterogeneous group of compounds that include local anesthetics, long chain alcohols, barbiturates, phencyclidine, ethidium, substance P, and insecticides (4). Noncompetitive antagonists used as photoaffinity probes of the AChR were instrumental in determining that the transmembrane ␣-helical M2 segment of each receptor subunit forms the lining of the ion channel pore (5-9). Under equilibrium binding conditions, the majority of NCAs interact preferentially with the AChR when acetylcholine sites are occupied by agonist, that is they bind preferentially to the desensitized state of the AChR. Photoaffinity labeling studies with NCAs have identified several unique binding loci within the channel of the AChR in the desensitized state (5-10). Examples of NCAs such as the local anesthetic tetracaine (11, 12) and the compound diazofluorene (13), which bind with appreciable (micromolar) affinity to the AChR channel when agonist is not bound to the receptor, which is in the resting state, are far less prevalent.
Abstract
The uncharged, photoreactive compound 3-trifluoromethyl-3-(m-[ 125 I]iodophenyl)diazirine ([ 125 I]TID) is another NCA that interacts with high affinity with the AChR channel in the resting state. TID is a potent noncompetitive antagonist of the AChR, inhibiting agonist-induced ion flux and binding with micromolar affinity to the channel in both the resting and desensitized states (14, 15) . TID is a unique compound in that there are two components to its photoincorporation into the AChR. First, TID is a potent NCA, and one component of [ 125 I]TID photoincorporation is localized to the channel-lining M2 segments of each receptor subunit. [
Abstract
125 I]TID photoincorporation into the M2 segment of each AChR subunit is inhibited in a concentration-dependent manner by TID itself as well as by other noncompetitive antagonists (14, 16, 17) . In addition to "specific" photoincorporation into amino acids in the M2 segments of each AChR subunit, [
Abstract
125 I]TID also photoincorporates into amino acids in the M1, M3, and M4 transmembrane segments of each receptor subunit. The extent of [ 125 I]TID photoincorporation into residues in the M1, M3, and M4 segments is not affected by the further addition of an excess of nonradioactive TID or other noncompetitive antagonists, that is the labeling is "nonspecific." Consistent with the hydrophobic nature of TID, it partitions extremely effectively into the lipid bilayer, and [
Abstract
125 I]TID photoincorporation into amino acids in the transmembrane segments M1, M3, and M4 is interpreted as indicating that these residues are situated at the lipid-protein interface of the .

corporation in the ␦M2 segment indicate a common binding locus for each TID analog. We conclude that relatively small additions to TID progressively reduce its ability to interact with the NCA site in the resting state channel. A model of the NCA site and resting state channel is presented.

Noncompetitive antagonists (NCAs) 1 of the nicotinic acetylcholine receptor (AChR) are by definition agents that block the permeability response of the receptor by binding to sites that are distinct from the agonist-binding sites (recent reviews include Refs. [1] [2] [3] . NCAs of the AChR are a large and structurally heterogeneous group of compounds that include local anesthetics, long chain alcohols, barbiturates, phencyclidine, ethidium, substance P, and insecticides (4) . Noncompetitive antagonists used as photoaffinity probes of the AChR were instrumental in determining that the transmembrane ␣-helical M2 segment of each receptor subunit forms the lining of the ion channel pore (5) (6) (7) (8) (9) . Under equilibrium binding conditions, the majority of NCAs interact preferentially with the AChR when acetylcholine sites are occupied by agonist, that is they bind preferentially to the desensitized state of the AChR. Photoaffinity labeling studies with NCAs have identified several unique binding loci within the channel of the AChR in the desensitized state (5) (6) (7) (8) (9) (10) . Examples of NCAs such as the local anesthetic tetracaine (11, 12) and the compound diazofluorene (13) , which bind with appreciable (micromolar) affinity to the AChR channel when agonist is not bound to the receptor, which is in the resting state, are far less prevalent.

The uncharged, photoreactive compound 3-trifluoromethyl-3-(m- [ 125 I]iodophenyl)diazirine ([ 125 I]TID) is another NCA that interacts with high affinity with the AChR channel in the resting state. TID is a potent noncompetitive antagonist of the AChR, inhibiting agonist-induced ion flux and binding with micromolar affinity to the channel in both the resting and desensitized states (14, 15) . TID is a unique compound in that there are two components to its photoincorporation into the AChR. First, TID is a potent NCA, and one component of [ 125 I]TID photoincorporation is localized to the channel-lining M2 segments of each receptor subunit. [ 125 I]TID photoincorporation into the M2 segment of each AChR subunit is inhibited in a concentration-dependent manner by TID itself as well as by other noncompetitive antagonists (14, 16, 17) . In addition to "specific" photoincorporation into amino acids in the M2 segments of each AChR subunit, [ 125 I]TID also photoincorporates into amino acids in the M1, M3, and M4 transmembrane segments of each receptor subunit. The extent of [ 125 I]TID photoincorporation into residues in the M1, M3, and M4 segments is not affected by the further addition of an excess of nonradioactive TID or other noncompetitive antagonists, that is the labeling is "nonspecific." Consistent with the hydrophobic nature of TID, it partitions extremely effectively into the lipid bilayer, and [ 125 I]TID photoincorporation into amino acids in the transmembrane segments M1, M3, and M4 is interpreted as indicating that these residues are situated at the lipid-protein interface of the AChR (18 -20) . I]iodophenyl)diazirine; TTDBA, TTDBAc, TTDBIBA, TTDBTMAc, TTD-BE 4Ј-(3-trifluoromethyl-3H-diazirin-3-yl)-2Ј-tributylstannylbenzyl (alcohol, acetate, isobutyric acetate, trimethyl acetate, benzoate); DMAP, 4-(dimethylamino)pyridine; PAGE, polyacrylamide gel electrophoresis; Tricine, N- [2- hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography; PTH, phenylthiohydantoin; TMB-8, 3,4,5-tri-Whereas TID binds with micromolar affinity to the AChR channel in both the resting and desensitized state (14) , [ 125 I]TID photoincorporates into amino acids in the M2 segment of each AChR subunit in the resting state with 10-fold greater efficiency than in the desensitized state. In the resting state, [ 125 I]TID reacts with homologous aliphatic residues in each M2 segment that are located 9 and 13 amino acid residues carboxyl-terminal to the conserved lysine residue at the amino terminus of the M2 region (e.g. ␦Leu-265 and ␦Val-269). These results provide important information about the NCA-binding site and suggest that the conserved leucine residues at position 9 (e.g. ␦Leu-265) may form a permeability barrier to the passage of ions in the resting state channel (16) .

The work presented here employs analogs of TID to examine further the molecular determinants of the NCA-binding site in the AChR channel in the resting state. The interaction of TID analogs with the NCA-binding site in the resting state channel were assessed in several ways as follows: 1) the ability of nonradioactive TID analogs to inhibit [ 125 ]TID indicate that each of the TID analogs binds to a single site in the resting state channel. We conclude that relatively small structural additions to TID result in a progressive reduction in the TID analogs ability to bind to and photolabel the NCA site in the resting state channel. The structure-activity studies with TID analogs and the AChR lead us to suggest a model for the structure of the resting state channel and the NCA-binding site.
EXPERIMENTAL PROCEDURES
Materials-AChR-rich membranes were isolated from Torpedo californica electric organ (21 1) were prepared by radioiododestanylation of the tin-based precursors 4Ј-(3-trifluoromethyl-3H-diazirin-3-yl)-2Ј-tributylstannylbenzyl alcohol (TTDBA) and 4Ј-(3-trifluoromethyl-3H-diazirin-3-yl)-2Ј-tributylstannylbenzyl benzoate (TTD-BE), respectively (10, 22, 23). The tin-based precursors were a generous gift from Dr. Josef Brunner of the Swiss Federal Institute of Technology (ETH), Zurich, Switzerland. Briefly, 45 nmol of TTDBA or TTD-BE in ethanol/toluene (1:1) were added to a 1-ml Reactivial and dried under nitrogen. The dried material was dissolved in 20 l of acetic acid, 2 mCi of Na 125 I was then added, and the iodination reaction initiated by the further addition of 5 l of peracetic acid. After 15 min the reaction was quenched by adding 3 l of NaI (100 mM) and 200 l of sodium bisulfite (10% (w/w)). The radioiodinated compound was extracted into 400 l of ethyl acetate (upper phase), which was removed and dried under nitrogen. The dried material was dissolved in 100 l of ethanol/toluene (1:1), applied to a Silica Gel 60 TLC plate (5 ϫ 20 cm), and developed with ether/hexane (1:9). ]TID-BE (R f ϭ 0.74) were scraped from the TLC plate, extracted with 1 ml of ethanol, dried under nitrogen, dissolved in 50 l of ethanol and stored at Ϫ20°C. Nonradioactive stocks of TIDBA and TID-BE were made in nearly identical fashion, and the concentrations were determined spectrophotometrically (⑀ 351 nm ϭ 450 (22)). The TID analogs TTDBAc, TTDBIBA, and TTDBTMAc were made by acylation of the alcohol function of the tin precursor TTDBA (22) . Briefly, 20 l of TTDBA (1.8 mol) was added to a 300-l Reactivial, the solvent removed by a stream of N 2 gas, and 4 mol of 4-(dimethylamino)pyridine (DMAP) and 100 l of anhydrous tetrahydrofuran added. Under anhydrous conditions, 50 mol of either acetic anhydride, isobutyric anhydride, or trimethylacetic anhydride were added, and the acylation reaction was allowed to proceed overnight at room temperature (in the dark). Following solvent evaporation and resuspension in ethanol, each of the tin precursors were purified by TLC (ether/hexane, 1:9), TTDBAc (R f ϭ 0.58), TTDBIBA (R f ϭ 0.72), and TTDBTMAc (R f ϭ 0.78). . For analytical labeling experiments, approximately 1 mg of AChR membranes were used and 10 mg (per condition) for preparative labelings. Incubations were performed in either glass test tubes or vials and under reduced lighting conditions. The samples were then irradiated with a 365 nm UV lamp (Spectroline EN-280L) for 7 min at a distance of less than 1 cm and centrifuged at 39,000 ϫ g for 1 h. Pellets were solubilized in electrophoresis sample buffer and subjected to SDS-PAGE (24) .
EXPERIMENTAL PROCEDURES
For analytical labelings 1.0-mm thick SDS-PAGE gels were used (1.5-mm thick for preparative labeling experiments) containing separating gels composed of 8% polyacrylamide, 0.33% bisacrylamide. Following electrophoresis, gels were stained with Coomassie Blue R-250 to visualize AChR subunit bands. Analytical gels were dried and exposed to Kodak X-Omat LS film with an intensifying screen at Ϫ80°C (1-3-day exposure). Preparative gels and analytical gels for which receptor subunits were to be subjected to proteolytic mapping were soaked in distilled water overnight, and the bands for ␣-, ␤-, ␥-, and ␦-subunits excised from the 8% gels. Proteolytic mapping of the sites of [ 125 I]TID analog photoincorporation within AChR subunits was performed according to the method of Cleveland et al. (25) and as described in detail in Blanton et al. (10) . For preparative gels, AChR subunits were eluted from the excised gel pieces into 15 ml of Elution Buffer (0.1 M NH 4 HCO 3 , 0.1% (w/v) SDS, 1% ␤-mercaptoethanol, pH 7.8) for 4 days at room temperature with gentle mixing. The gel suspensions were then filtered through Whatman No. 1 paper and concentrated using a Centriprep-10 (Amicon). Excess SDS was removed by acetone precipitation (ϳ85% acetone at Ϫ20°C for 12 h).
EXPERIMENTAL PROCEDURES
For I ]TIDBIBA (10 mg of AChR-rich membranes per labeling condition) under conditions that favor either the resting state or the desensitized state (i.e. in the presence of the agonist carbamylcholine). AChR subunits were resolved on 1.5-mm thick 8% acrylamide gels, and the ␦-subunit was isolated as described above. For digestion with trypsin, acetone-precipitated ␦-subunits were resuspended in approximately 300 l of 0.1 M NH 4 HCO 3 , 0.02% (w/v) SDS, 0.5% Genapol C-100, pH 7.8 (1-2 mg/ml protein). Trypsin was added at a 20% (w/w) enzyme to substrate ratio, and the digestion was allowed to proceed 4 -5 days at room temperature. A small aliquot of each sample was electrophoresed on an analytical (1.0-mm thick) 16.5% T, 6% C Tricine SDS-PAGE gel with at least one reference lane containing prestained low molecular weight protein standards (Life Technologies, Inc). The Tricine gel was stained, destained, and dried for autoradiography. The bulk of the ␦-subunit tryptic digests were solubilized in electrophoresis sample buffer and submitted to preparative (1.5-mm thick) scale 16.5% T, 6% C Tricine gel analysis (10, 13, 16) . Preparative Tricine gels were stained, destained, and soaked in water overnight. By using the autoradiograph from the analytical Tricine gel in conjunction with the prestained protein standards, an approximately 5-kDa band known to contain the M2 segment of the ␦-subunit (␦T-5K (13, 16)) was excised from the gel and eluted into 4 ml of Elution Buffer for 4 days at room temperature. The ␦T-5K fragment was further purified by reverse-phase HPLC using a Brownlee Aquapore C 4 column (100 ϫ 2.1 mm) with Solvent A (0.08% trifluoroacetic acid in water) and Solvent B (0.05% trifluoroacetic acid in 60% acetonitrile, 40% 2-propanol). A nonlinear elution gradient was employed (25-100% Solvent B in 80 min), and the elution of peptides was monitored by the absorbance at 210 nm. Collected fractions were counted for radioactivity, and the peak protein/radioactivity-containing fractions were pooled, dried by vacuum centrifugation, and resuspended in 20 l of 0.1 M NH 4 HCO 3 , 0.1% (w/v) SDS, pH 7.8, for protein radiosequence analysis.
EXPERIMENTAL PROCEDURES
Sequence Analysis-Amino-terminal sequence analysis was performed on a Beckman Instruments (Porton) model 20/20 protein sequencer using gas phase cycles (Texas Tech University Biotechnology Core Facility). Peptide aliquots (25 l) were immobilized on chemically modified glass fiber disks (Beckman Instruments), which were used to improve the sequencing yields of hydrophobic peptides. Approximately 20% of the release PTH-derivatives were separated by an on-line PTHderivative analyzer, and approximately 80% was collected for determination of released 125 I cpm by ␥-counting of each sample for 20 min. Initial yield (I o ) and repetitive yield (R) were calculated by nonlinear least squares regression of the observed release (M) for each cycle (n):
EXPERIMENTAL PROCEDURES
n (PTH-derivatives of Cys and His were omitted from the fit). from the dried polyacrylamide gel and determining the amount of 125 I cpm present by ␥-counting. 3B ) there appears to be less of a difference in the extent of photoincorporation into each AChR subunit when the receptor is labeled in the absence of agonist (Fig. 3B , Ϫ lane) compared with the presence of agonist (Fig. 3B, ϩ lane) . Finally, for the TID analog [ 125 I]TIDBTMAc, when the photolabeling is done in the absence of agonist (Fig. 3C, Ϫ lane) , the extent of photoincorporation into each AChR subunit is approximately equal, that is there is not a greater extent of labeling in the ␥-subunit relative to the ␣-, ␤-, or ␦-subunits as observed for TID, TIDBA, TIDBAc, and TIDBIBA. Furthermore, the extent of photoincorporation into each AChR subunit is the same whether the photolabeling is done in the absence of agonist (Fig. 3C, Ϫ lane) or in the presence of agonist (Fig. 3C, ϩ lane) .
RESULTS

TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
Differences in the extent of photoincorporation by TID analogs into AChR subunits were quantified by ␥-counting of excised gel bands. In the absence of agonist, the 4-fold greater magnitude of photoincorporation into the ␥-subunit relative to the other AChR subunits by [ 125 I]TID is maintained for the Fig. 1 (2nd to 7th) . AChR-rich membranes were then irradiated at 365 nm for 7 min, and polypeptides were resolved by SDS-PAGE. A, shown is the corresponding autoradiograph of the gel containing the competition labeling experiment [ Table I . TIDBA (q), TIDBAc (OE), TIDBIBA (), TIDBTMAc (ࡗ), TID-BE (f).
TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
analogs 
TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
I]TID analog, in each case the labeled AChR ␣-subunit (Ϫ and ϩ carbamylcholine labeling condition) was partially digested using S. aureus V8 protease under Cleveland gel conditions (10, 20, 25) . Cleveland gel analysis of the AChR ␣-subunit generates four large, non-overlapping fragments of the ␣-subunit allowing the distribution of 125 I cpm within the subunit to be determined. For each of the [
TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
125 I]TID analogs, photoincorporation in the AChR ␣-subunit was found to be restricted to two V8 protease fragments, ␣V8 -20 (Ser-173-Glu-338) and ␣V8 -10 (Asn-339 -Gly-437; data not shown). The stretch of the primary sequence of the ␣-subunit contained within the V8 protease fragment ␣V8 -20 includes the transmembrane segments M1, M2, and M3; the fragment ␣V8 -10 contains the transmembrane segment M4. The extent of [
TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
125 I]TID analog photoincorporation into the V8 protease fragments ␣V8 -20 and ␣V8 -10 for AChRs labeled in the absence and presence of agonist is summarized in Table III (Table III) .
TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
When the distribution of 125 I cpm within the ␣-subunit was determined for AChRs labeled with each of the [
TID Analog Inhibition of [ 125 I]TID Photoincorporation into the AChR Channel in the Resting
125 I]TID analogs in the desensitized state, photoincorporation into both V8 protease fragments as a percentage of the total are approximately equivalent (Table III) . In addition, although not presented in Table III I ]TID analog photoincorporation was restricted to two V8 protease fragments: ␣V8 -20 (Ser-173-Glu-338) and ␣V8 -10 (Asn-339 -Gly-437) and which contain the membrane-spanning segments M1-M3 and M4, respectively. ␣V8 -20 contains the channel-lining M2 segment, and ␣V8 -10 contains the M4 segment which is situated at the lipid-protein interface (10, 20 (10, 19, 20) . For the fragment ␣V8 -20, there is photoincorporation into amino acids in the transmembrane segments M1 and M3, and for ␣V8 -10, photoincorporation into amino acids within M4. It was further concluded that these amino acids are in contact with lipid, with the AChR transmembrane segments M1, M3, and M4 comprising the lipid-protein interface of the receptor. The simplest interpretation of the ␣-subunit mapping results for each [ 125 I]TID analog is as follows: 1) that agonist-sensitive photoincorporation within the V8 fragment ␣V8 -20 reflects labeling of amino acids in the ␣M2 segment; 2) that photoincorporation into the V8 fragment ␣V8 -10 and the bulk of photoincorporation into ␣V8 -20 for desensitized state AChRs reflects labeling of amino acid residues in the transmembrane segments M1, M3, and M4 that are in contact with lipid.
Inhibition of [ 125 I]TID Analog Photoincorporation into the AChR Channel in the Resting State by the Noncompetitive Antagonists TMB-8 and Tetracaine-To confirm that the differences in the agonist sensitivity of [
125 I]TID analog photoincorporation into AChR subunits is truly a reflection of differences in the extent of photoincorporation into the resting state channel, we tested the ability of NCAs that bind to the AChR channel in the resting state to inhibit [ 125 I]TID analog photoincorporation into receptor subunits. 2 The local anesthetic tetracaine binds with high affinity (K eq ϭ 0.3 M) to a single site in the resting state AChR, and this site has been localized by photoaffinity labeling to the M2 segment of each receptor subunit that together comprise the resting state channel (11, 12) . TMB-8 (3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester) binds with equal affinity to the AChR channel in both its resting and desensitized states and has been shown to inhibit competitively [ (Fig. 4, 7th lane) , the level of [ 125 I]TIDBIBA photoincorporation into AChR subunits is reduced nearly to that observed in the presence of agonist (Fig. 4, 8th lane) (Table IV) . In contrast, whereas 50 M TMB-8 reduced [
Inhibition of [ 125 I]TID Analog Photoincorporation into the AChR Channel in the Resting State by the Noncompetitive Antagonists TMB-8 and Tetracaine-To confirm that the differences in the agonist sensitivity of [
125 I]TIDBIBA photoincorporation into the ␥-subunit by greater than 98%, it had no effect (less than 5% reduction) on the extent of photoincorporation of [ 125 I]TIDBTMAc into the ␥-subunit ( Fig. 4B; is the easiest M2 segment to isolate, ␥M2 being the most difficult to isolate and is prone to irreversible aggregation (13); (c) because the amino acids photolabeled by [ 125 I]TID-BE in the ␦M2 segment have also been determined. AChR-rich membranes (10 mg per condition) were labeled with [
Inhibition of [ 125 I]TID Analog Photoincorporation into the AChR Channel in the Resting State by the Noncompetitive Antagonists TMB-8 and Tetracaine-To confirm that the differences in the agonist sensitivity of [
125 I]TIDBIBA (3 M) in the absence and presence of carbamylcholine. The ␦-subunits isolated from each condition were digested with 20% (w/w) trypsin for 4 days. The digests were resolved by Tricine SDS-PAGE, and a 5-kDa fragment (␦T-5K) known to contain the M2-M3 region (16, 13) was isolated from the gel as described under "Experimental Procedures." The ␦T-5K fragment was further purified by reversed-phase HPLC (Fig. 5A) , and for each labeling condition the majority of 125 I cpm eluted in a peak centered at 98% solvent B. HPLC fractions 36 -39, for ␦T-5K isolated from resting state AChRs (Fig. 5A, q) , were pooled and subjected to amino-terminal amino acid sequence analysis (Fig.  5B) . Sequence analysis revealed the presence of a single sequence, present at a 10-fold or greater level than any secondary sequence, beginning at ␦Met-257 at the amino terminus of ␦M2. The primary site of 125 I release occurred in cycle 13 with a small amount of release also present in cycle 9 (Fig. 5B, q) . These results indicate that within the ␦M2 segment and in the resting state AChR, [ 125 I]TIDBIBA photoincorporates into ␦Leu-265 (0.71 cpm/pmol) and ␦Val-269 (5.2 cpm/pmol). These same two residues are also the primary sites of [ HPLC fractions 36 -39 for ␦T-5K isolated from desensitized state AChRs (Fig. 5A, E) were also pooled and subjected to ]TIDBIBA-labeled AChR ␦-subunit isolated from AChRs labeled in the resting state (q) and desensitized state (E) were further digested in solution with trypsin, and an approximately 5-kDa (␦T-5K) fragment was isolated from a 1.5-mm thick 16.5% T, 6% C Tricine gel (see "Experimental Procedures"). The labeled material was further purified by reverse-phase HPLC (A) on a Brownlee Aquapore C 4 column (100 ϫ 2.1 mm) as described under "Experimental Procedures." The elution of peptides was monitored by absorbance at 210 nm (solid line) and elution of 125 I by ␥-counting of each 500 l fraction (q, E). For each condition, HPLC fractions 36 -39 were pooled and subjected to automated sequential Edman degradation. B, shown is the radiosequencing profile for the ␦T-5K fragment that contains the M2 segment of the ␦-subunit isolated from AChRs labeled in the resting state. Eighty percent of each cycle of Edman degradation was analyzed for released 125 I (q) and 20% for released PTH-derivatives (Ⅺ) with the dashed line corresponding to the exponential decay fit of the amount of detected PTH-derivatives. A primary peptide was detected beginning at Met-257 of the ␦-subunit (initial yield, 130 pmol; repetitive yield, 89.9%; 15,495 cpm loaded on sequencing filter; 6,477 cpm remaining after 20 cycles). The amino acid sequence of the peptide is shown above B with the solid line indicating the limits of the M2 region.
Inhibition of [ 125 I]TID Analog Photoincorporation into the AChR Channel in the Resting State by the Noncompetitive Antagonists TMB-8 and Tetracaine-To confirm that the differences in the agonist sensitivity of [
amino-terminal amino acid sequence analysis (Fig. 6) . As with ␦T-5K isolated from resting state AChRs, sequence analysis revealed the presence of a single sequence, present at a ϳ10-fold greater level than any secondary sequence. The primary sequence began at ␦Met-257, the amino terminus of the ␦M2 segment. The 125 I cpm release profile was somewhat complex but indicated a small amount of release occurring in cycles 6, 9, 13, and possibly cycle 18 (Fig. 6, q) . Release in these cycles corresponds to [ 125 I]TIDBIBA photoincorporation into ␦Ser-262 (0.11 cpm/pmol), ␦Leu-265 (0.21 cpm/pmol), ␦Val-269 (0.19 cpm/pmol), and ␦Thr-265 (0.10 cpm/pmol). These same amino acids were determined to be the primary sites of [
Inhibition of [ 125 I]TID Analog Photoincorporation into the AChR Channel in the Resting State by the Noncompetitive Antagonists TMB-8 and Tetracaine-To confirm that the differences in the agonist sensitivity of [
125 I]TID photoincorporation into ␦M2 in the desensitized state AChR (16) , indicating that TID and TIDBIBA bind to the same locus in the desensitized state channel. In contrast, based upon [ 125 I]TID-BE photoincorporation into ␤Leu-257, ␤Val-261, and ␤Leu-264 in the ␤M2 segment, it was concluded that compared with TID, TID-BE binds to a unique but overlapping site in the desensitized state channel. The binding locus for TID-BE appears to be located slightly higher in the channel, toward the extracellular side of the membrane (10) .
Inhibition of [ 125 I]TID Analog Photoincorporation into the AChR Channel in the Resting State by the Noncompetitive Antagonists TMB-8 and Tetracaine-To confirm that the differences in the agonist sensitivity of [
Finally, the interaction of TIDBIBA with the AChR channel in the desensitized state was further assessed by examining the effect of TIDBIBA on the binding of 
DISCUSSION
The goal of this work was to gain insight into the molecular determinants of NCA binding to the channel of the AChR in the resting state. Our strategy for achieving this goal involved employing a series of analogs of the potent AChR NCA, [ 125 I]TID. The binding site for TID in the AChR channel in the resting state has been well characterized. TID binds with unitary stoichiometry and micromolar affinity to the resting state channel, photoincorporating into homologous aliphatic residues at positions 9 and 13 (e.g. ␤Leu-257 and ␤Val-261) of each M2 segment (14 -16) . We assessed the ability of analogs of TID (Fig. 1) (Table IV) . We next converted the alcohol function of TIDBA into either an acetate, isobutyl acetate, or trimethyl acetate group (TIDBAc, TIDBIBA, or TIDBTMAc, respectively; Fig. 1 ). For these TID analogs there was a progressive rightward shift in the concentration-response curves for inhibition of [ 125 I]TID photoincorporation into AChR subunits in the resting state, resulting in 1.5-, 5.25-, and 11-fold decreases in potencies (Table I) . This decrease in the ability of a given concentration of TID analog to inhibit [ 125 I]TID photoincorporation into the resting state channel parallels a decrease in the agonist sensitivity of [ 125 I]TID analog photoincorporation into AChR subunits (Fig. 3 and Table II). For [ 125 I]TID, the addition of agonist (desensitized state of AChR) results in an ϳ95% decrease in the extent of photoincorporation into the ␥-subunit. This decrease in photoincorporation into the ␥-subunit (as well as the ␣-, ␤-, and ␦-subunits) is a result of decreased efficiency of [ 125 I]TID photoincorporation into amino acids in the M2 segment that results from an agonist-induced change in the structure of the 3 M. P. Blanton, unpublished data. 125 I]TID-BIBA-photolabeled AChR ␦-subunit isolated from desensitized state AChRs was further digested in solution with trypsin, and an approximately 5-kDa (␦T-5K) fragment was isolated from a 1.5-mm thick 16.5% T, 6% C Tricine gel and further purified by reverse-phase HPLC (Fig.  5A ). HPLC fractions 36 -39 for ␦T-5K isolated from desensitized state AChRs (Fig. 5A , E) were pooled and subjected to automated sequential Edman degradation. Shown is the radiosequencing profile for the ␦T-5K fragment that contains the M2 segment of the ␦-subunit isolated from desensitized state AChRs. Eighty percent of each cycle of Edman degradation was analyzed for released 125 I (q) and 20% for released PTHderivatives (Ⅺ) with the dashed line corresponding to the exponential decay fit of the amount of detected PTH-derivatives. A primary peptide was detected beginning at Met-257 of the ␦-subunit (initial yield, 112 pmol; repetitive yield, 92.6%; 8,417 cpm loaded on sequencing filter; 2,805 cpm remaining after 20 cycles). The amino acid sequence of the peptide is shown above the panel with the solid line indicating the limits of the M2 region.
DISCUSSION
channel and the NCA site (16 (Table II) . The simplest interpretation of these results is that there is a progressive reduction in the ability of each TID analog as cited to interact with the NCA site in the resting state channel. In other words, each consecutive TID analog has a reduced affinity for binding to the resting state channel and a reduced ability to photoincorporate efficiently into amino acids within the NCA site. There are several lines of evidence that support this interpretation. First, there is both direct and indirect evidence that the TID analogs are binding to a common site within the resting state channel. In resting state AChR, [ 125 I]TID photoincorporates into the ␥-subunit at a 4-fold greater level than into the ␣-, ␤-, or ␦-subunits. The greater extent of photoincorporation of [ 125 I]TID into the ␥-subunit relative to the ␣-, ␤-, or ␦-subunits is a consequence of highly efficient photoincorporation into a single amino acid, Ile-264 (position 13) of the ␥M2 segment (16) . This 4-fold greater photoincorporation into the ␥-subunit is maintained for photoincorporation into the AChR subunits by [ 125 I]TIDBIBA photoincorporation in ␦M2), the additional bulk of TIDBTMAc is then in approximate register with ␦Leu-265, located one turn of the ␦M2 ␣-helix lower in the channel. Structural studies of the AChR suggest that in the resting state channel ␦Leu-265 as well as the leucine residue at this position (position 9) in the M2 segment in each of the other subunits are in close proximity to one another and together form a restriction, or gate, to ion permeation (16, 28, 29) . Whereas the channel-lining M2 segments are predominantly ␣-helical, structural studies (28, 29) suggest that each M2 helix has a central kink around position 9 (e.g. ␦Leu-265). The kink in the M2 helix results in a narrowing of the channel at this position, bringing the side chains of each conserved leucine residue in close apposition to one another (27) . The narrowing of the channel at this position and the close apposition of the aliphatic side chains of each leucine residue in all likelihood provides an ideal binding site for uncharged NCAs such as TID as well as diazofluorene (13) . On the other hand, the restriction in the lumen of the channel is also likely to present a progressively greater amount of steric hindrance to the binding of each of the TID analogs, TIDBA, TIDBAc, TIDBIBA, TIDBTMAc, and TID-BE, in which an increasingly bulkier substituent has been added to the TID parent compound. For TIDBTMAc and TID-BE the affinity for the NCA site in the resting state channel and the efficiency of photoincorporation into M2 residues are so reduced that labeling of the resting state channel is not apparent at the level of the intact subunit. However, close examination of the sites of Finally, a recent study (12) determining the sites of [ 3 H]tetracaine photoincorporation in the M2 segments of the AChR in the resting state suggest that the restriction in the lumen of the channel (at position 9 of each M2 segment) is somewhat flexible. [ 3 H]Tetracaine photoincorporates into Ile-247 (position 5), Leu-251 (position 9), and Val-255 (position 13) in the ␣M2 segment. [ 3 H]Tetracaine photoincorporation into Ile-247 in ␣M2 places at least a portion of the tetracaine molecule below the apparent restriction in the lumen of the channel at position 9. This fact and that the kinetics of tetracaine binding to the resting state AChR are characterized by a very low association rate constant led the authors (Gallagher and Cohen (12)) to conclude that the restriction in the pore of the resting state channel relaxes somewhat in order to accommodate the binding of tetracaine. The authors further suggest that the dimethylamino group of tetracaine interacts with Ser-248 (position 6) in ␣M2 providing energetic stability to tetracaine binding to the resting state channel. Synthesis of additional TID analogs, including analogs that contain a dimethylamino substituent, as well molecular modeling studies will undoubtedly yield a more complete picture of the NCA site in the resting state channel and of the molecular determinants of NCA binding.

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