Introduction

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The nicotinic acetylcholine receptor (nAChR) of vertebrate skeletal muscle and Torpedo species electric organ is a well characterized ligand-gated ion channel that exists in at least three interconvertible conformations: the resting (closed channel) state, the open channel state, and the agonist bound, nonconducting desensitized state (Hucho et al., 1996; Corringer et al., 2000). The nAChR is a pentamer of four homologous subunits (₂) with each subunit containing four hydrophobic transmembrane segments (M1-M4). The M2 segments of each subunit are -helical and associate at the central axis of the nAChR to form the ion channel (Hucho et al., 1986; Imoto et al., 1988; Charnet et al., 1990; Revah et al., 1990; Imoto et al., 1991), with additional contributions from N-terminal residues of the M1 segments (Zhang and Karlin, 1997). The M3 and M4 segments contribute to the lipid-protein interface (Blanton and Cohen, 1994).

Many positively charged noncompetitive antagonists (NCAs), compounds that inhibit nAChR function by binding to sites other than the agonist binding site, bind within the lumen of the ion channel (for review, see Arias, 1998). Mutations in M2 affect the potencies of aromatic amine NCAs, including QX-222 (Charnet et al., 1990) and phencyclidine (Eaton et al., 2000), whereas mutations in M1 affect quinacrine potency (Tamamizu et al., 1995). For Torpedo species nAChRs equilibrated with agonist (i.e., in the desensitized state), the NCAs [³H]chlorpromazine and [³H]triphenylmethylphosphonium photoaffinity-labeled residues toward the N-terminal (cytoplasmic) end of each M2 segment (residues M2-6 and M2-9, with reference to the conserved Lys at the N-terminal end of each M2 segment) (Giraudat et al., 1986, 1987, 1989; Hucho et al., 1986; Revah et al., 1990), whereas [³H]meproadifen mustard reacted with Glu-262 (M2-20) at the extracellular end (Pedersen et al., 1992). In the absence of agonist (i.e., in the closed channel state), [³H]tetracaine was specifically photoincorporated into amino acids in the middle of each M2 segment (M2-9 and M2-13) (Gallagher and Cohen, 1999). In contrast, in the open channel state, [³H]quinacrine azide photolabeled amino acids within the M1 segment (DiPaola et al., 1990). Affinity labeling studies also have shown that uncharged NCAs bind within the nAChR ion channel. In the absence of agonist, [¹²⁵I] 3-(trifluoromethyl)-3-(m-iodophenyl)diazirine (TID) specifically labeled the same amino acids as [³H]tetracaine (M2-9 and -13 of each subunit), whereas in the desensitized state [¹²⁵I]TID labeled residues deeper in the pore (M2-2 and -6) (White and Cohen, 1992). In the absence of agonist, [³H]diazofluorene selectively labeled amino acids in -subunit (M2-9 and -13) and in the -subunit (M2-13), with the labeling in the desensitized state also shifted to residues closer to the cytoplasmic end (M2-6, M2-9 and -10) (Blanton et al., 1998).

[³H]Histrionicotoxin (HTX) and [³H]phencyclidine (PCP) are well characterized NCAs that have each been shown to bind reversibly to a single site in the Torpedo species nAChR (Heidmann et al., 1983). [³H]HTX binds with similar high affinity (K_D = ~0.3 µM) to nAChRs in the resting and desensitized states, whereas [³H]PCP binds with K_D values of 1 µM to the desensitized state and ~6 µM to the resting state. They serve as useful probes of the NCA site in the nAChR because they bind relatively weakly to the ACh sites, and they seem to interact with fewer low-affinity sites in the Torpedo species nicotinic postsynaptic membrane than other, more lipophilic NCAs. Although these compounds are assumed to bind within the ion channel, the binding site for neither NCA has been directly identified by affinity labeling. HTX and PCP bind in a mutually exclusive manner with each other and with other aromatic amine NCAs. In the desensitized state, PCP and HTX bind competitively with [³H]meproadifen mustard (Dreyer et al., 1986), a compound that seems to bind at the extracellular end of the ion channel domain (M2-20), and also with [³H]chorpromazine (Heidmann et al., 1983), a compound that binds lower in the channel domain (M2-6 and -9). In the absence of agonist, PCP and HTX bind competitively with [³H]tetracaine (Middleton et al., 1999), a result suggesting that the three ligands bind in a mutually exclusive manner to a common site at M2-9 and M2-13. However, in the absence of agonist, PCP and HTX interact allosterically with TID, a compound that binds to the same site as [³H]tetracaine. When evaluated at the level of nAChR subunits, PCP binding to its high-affinity site inhibited specific [¹²⁵I]TID photoincorporation in the -, -, and -subunits by only 20 to 30%, whereas it increased labeling in the -subunit (White and Cohen, 1988; Ryan et al., 2001). TID at 100 µM did not effect [³H]PCP binding, and it increased the K_D value of [³H]HTX only 5-fold (White et al., 1991).

To further characterize drug interactions within the ion channel, we have mapped, at the amino acid level, the binding site for [¹²⁵I]TID in the ion channel in the presence of tetracaine, PCP, or HTX. We wanted to test the hypothesis that tetracaine would reduce [¹²⁵I]TID photoincorporation in the M2 ion channel domain in a manner consistent with mutually exclusive binding. We also wanted to determine whether the binding site for [¹²⁵I]TID in the ion channel was shifted in the presence of either HTX or PCP.

	Experimental Procedures

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Materials. nAChR-rich membranes were isolated from electric organs of Torpedo species (Winkler Enterprises, San Pedro, CA) as described elsewhere (Pedersen et al., 1986). The membranes used contained 1 to 2 nmol of [³H]ACh binding sites per milligram of protein. Endoproteinase Lys-C (EndoLys-C) was from Roche Molecular Biochemicals (Indianapolis, IN), 1-tosylamido-2-phenylethychlormethyketone-treated trypsin was from Worthington Biochemical (Freehold, NJ), and Staphylococcus aureus glutamyl endopeptidase (V8 protease) was from ICN Biomedical Inc. (Cosa Mesa, CA). Prestained low molecular mass gel standards were from Life Technologies (Gaithersburg, MD): ovalbumin (43 kDa), carbonic anhydrase (29 kDa), -lactoglobulin (18 kDa), lysozyme (14 kDa), bovine trypsin inhibitor (6 kDa), and insulin (2.8 kDa). Genapol C-100 (10%) and trifluoroacetic acid were from Pierce (Rockford, IL). 1-Azidopyrene was bought from Molecular Probes (Eugene, OR). [¹²⁵I]TID (10 Ci/mmol) was obtained from Amersham Pharmacia Biotech (Piscataway, NJ), and [³H]tetracaine (48 Ci/mmol) was prepared by tritium reduction (PerkinElmer Life Science Products, Boston, MA) of 3,5-dibromotetracaine as described previously (Gallagher and Cohen, 1999). PCP was from Applied Science (State College, PA) and HTX was kindly provided by Dr. Y. Kishi (Harvard University, Cambridge, MA). Nonradioactive TID was a gift from Dr. S. Husain (Massachusetts General Hospital, Boston, MA) and was repurified by silica gel chromatography (100% hexane elution) and stored in 100% ethanol after the hexane was removed by rotary evaporation. Purity and concentration of the TID was assessed by silica thin-layer chromatography and UV absorption at 365 nm (Brunner and Semenza, 1981). Structures of the drugs are shown in Fig. 1.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Structures of nAChR noncompetitive antagonists.

[³H]Tetracaine Binding. The equilibrium binding of [³H]tetracaine (10 nM) to nAChR-rich membranes in Torpedo species physiological saline (250 mM NaCl, 5 mM KCl, 3 mM CaCl₂, 2 mM MgCl₂, 5 mM NaPi, pH 7.0) was measured by a filtration assay (Middleton et al., 1999). Membrane suspensions (400 nM ACh sites) were incubated in the dark for 30 min at room temperature with [³H]tetracaine and various concentrations of nonradioactive TID before filtration, and nonspecific binding was defined as the amount of [³H]tetracaine bound in the presence of 100 µM nonradioactive tetracaine.

[¹²⁵I]TID Photolabeling. Photolabeling on an analytical scale was used to quantify [¹²⁵I]TID photoincorporation into nAChR subunits in the presence of tetracaine at concentrations from 1 µM to 1 mM. nAChR-rich membranes (100 µl, 2 mg of protein/ml) were incubated in the dark in microtiter plates with [¹²⁵I]TID (6.5 µM) and nonradioactive tetracaine for 30 min. The suspensions were then photolyzed for 30 min at 365 nm (model EB-280C; Spectroline, Westbury, NY) at a distance of 6 cm. Sample buffer was then added directly to the membrane suspensions, which were then separated by SDS-PAGE gel on an 8% acrylamide gel. The polypeptides on the gel were visualized by Coomassie blue stain, and the [¹²⁵I]TID incorporation in the gel was detected by autoradiography or by use of a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The nAChR -subunit ¹²⁵I incorporation was quantified using the ImageQuaNT software, with background levels calculated locally.

Proteolytic Digestion and Fragment Purification. The goal in these experiments was to characterize the effect of noncompetitive antagonists on [¹²⁵I]TID incorporation into the M2 hydrophobic segments. Therefore, the techniques used to generate the [¹²⁵I]TID-labeled fragments used the digestion conditions previously optimized (White and Cohen, 1992; Gallagher and Cohen, 1999) to produce fragments beginning at the N termini of the M2 segments. Briefly, solutions (1 mg of protein/ml) of V8-20 fragments and intact -subunit in 15 mM Tris, pH 8.1, 0.1% SDS were digested for 3 to 5 days with 4 U/ml of EndoLys-C, whereas the -subunit (approximately 1 mg/ml) in 100 mM NH₄HCO₃, pH 7.8/0.02% SDS/0.5% Genapol C-100 was digested with trypsin [3:2 (w/w), protein/trypsin] for 3 to 7 days. The V8-20 and -subunit digests were fractionated on 16.5% T/3% C Tricine gels (Schagger and von Jagow, 1987), and the -subunit digests were resolved on a 16.5%T/6%C Tricine gel to obtain higher resolution. Regions of the gels that were expected to contain peptides beginning at the N termini of the M2 segments were identified based upon the migration of prestained molecular mass markers and the pattern of 1-AP fluorescence (Gallagher and Cohen, 1999). Nonfluorescent regions at 8 and 7 kDa were excised from the V8-20 and -subunit digests, respectively, and a fluorescent band at 10 kDa was excised from the -subunit digest. Material was eluted from the bands, and the eluates were concentrated to 0.3 ml in Centricon-3 microconcentrators (Amicon). The concentrates were then fractionated by reversed phase HPLC as a second purification step that also removed the majority of the SDS. Based upon the distribution of ¹²⁵I, fractions were pooled, dried by vacuum centrifugation, and then resuspended in 25 µl of 100 mM NH₄HCO₃/0.05% SDS.

Sequence Analysis. The purified samples were applied to chemically modified glass fiber filters (Beckman, Palo Alto, CA) that were first washed with distilled water and methanol to remove impurities that inhibit binding of hydrophobic peptides. The purified peptides were fixed to the filters by delivery of gas trifluoroacetic acid for 4 min and then the SDS was removed from the filters by washing them for 5 min with ethyl acetate. The peptides were sequenced on an ABI 477 protein sequencer (Applied Biosystems, Foster City, CA) with gas phase sequencing cycles. During Edman degradation, two thirds of each sample was sent to a fraction collector for ¹²⁵I gamma counting and one third was injected on an amino acid analyzer (ABI 120A) to identify the PTH-amino acid. Picomoles of each PTH amino acid were calculated from the peak height on the analyzer chromatogram (ABI 610A Data Analysis Program version 1.2.1). The background-subtracted yields (excluding serines and cysteines) of each sequencing cycle were fit to the equation I(n) = I₀ × Rⁿ, where I(n) is picomoles of PTH amino acid at cycle n, I₀ is the initial peptide quantity in picomoles, and R is the sequencer repetitive yield. The efficiency of [¹²⁵I]TID incorporation into residue n, expressed as cpm_n per picomole (I_n), was calculated by [cpm_n  cpm_{n  1}]/2 × I_n with the factor of 2 in the denominator because at each cycle the volume counted was twice the volume injected into the amino acid analyzer. We employed a single factor ANOVA test (Microsoft Excel) to determine whether tetracaine, PCP, or HTX induced significant differences in the percentage of change of [¹²⁵I]TID labeling efficiencies of M2-9, M2-13, M2-9, and M2-13. We also used a two-tailed t test (Microsoft Excel; Microsoft, Redmond, WA) to determine whether there were significant differences in the ratios of ¹²⁵I incorporation in M2-9 and M2-13 within a single subunit.

	Results

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In initial experiments, we examined the effects of tetracaine on the photoincorporation of [¹²⁵I]TID into nAChR-rich membranes as well as the effects of TID on the reversible binding of [³H]tetracaine (Fig. 2). nAChR-rich membranes (1.5 µM ACh sites) were equilibrated with 6.5 µM [¹²⁵I]TID and various concentrations of tetracaine, and after photolysis the polypeptides were separated by SDS-PAGE. The gel was stained with Coomassie blue to allow identification of nAChR subunits and to verify that the lanes contained similar amounts of protein, and the distribution of ¹²⁵I was determined by PhosphorImaging (Fig. 2A). Consistent with previous results (White and Cohen, 1988), [¹²⁵I]TID was photoincorporated into each nAChR subunit, with the -subunit labeled most efficiently. (A prominently labeled -subunit fragment migrating near 45 kDa was also noted.) Tetracaine reduced ¹²⁵I incorporation in each nAChR subunit in a dose-dependent manner, whereas it had no effect on the ¹²⁵I incorporation in the -subunit of the Na⁺/K⁺-ATPase, which was labeled at lower efficiency than any of the nAChR subunits in the absence of tetracaine. When the concentration dependence of tetracaine inhibition of ¹²⁵I incorporation in nAChR -subunit was quantified (Fig. 2B), the data were fit by a single site model with an IC₅₀ value of 3 µM, and tetracaine reduced subunit labeling by ~90%. The IC₅₀ value was the same as for its inhibition of [³H]HTX binding and, as expected, for tetracaine binding to its high-affinity site in the nAChR M2 ion channel domain (Gallagher and Cohen, 1999; Middleton et al., 1999). Nonradioactive TID also inhibited the reversible binding of [³H]tetracaine (Fig. 2B) with a concentration dependence characterized by an IC₅₀ value of 3 µM. At high concentrations, TID inhibited at least 95% of specifically bound [³H]tetracaine. These results contrasted with the interactions between TID and PCP or HTX (White and Cohen, 1988; White et al., 1991). TID did not inhibit [³H]PCP binding in the absence of agonist, and PCP reduced [¹²⁵I]TID photolabeling of nAChR -subunit by less than 30%.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Mutual inhibition of tetracaine and TID binding to the nAChR. A, suspensions of nAChR-rich membranes (2 mg/ml) were equilibrated with 6.5 µM [125I]TID and tetracaine (lanes 1-8: 0, 1, 3, 10, 30, 100, 300, 1000 µM) and then photolyzed at 365 nm. The membrane polypeptides were then fractionated by SDS-PAGE and visualized by Coomassie blue staining (data not shown). 125I incorporation in the polypeptides was visualized by phosphoimaging. B, concentration dependence of tetracaine inhibition of [125I]TID incorporation into nAChR -subunit () and of TID inhibition of the equilibrium binding of [3H]tetracaine (). For the experiment in A and two similar experiments 125I incorporation in the nAChR -subunit was quantified using ImageQuaNT software (, mean ± S.D.). The concentration dependence of tetracaine inhibition was fit to a single-site model with an IC50 value of 3.1 ± 0.2 µM and a maximal inhibition of 92 ± 1%. The equilibrium binding of [3H]tetracaine (10 nM) to nAChR-rich membranes (200 nM nAChR) was determined by a filtration assay, with the nonspecific binding defined as the amount of 3H retained in the presence of 100 µM tetracaine. The percentage of specifically bound [3H]tetracaine [f(x)] was calculated at each concentration of TID (x), and the data points are the average and standard deviations of three experiments. The concentration dependence of inhibition of [3H]tetracaine binding (or [125I]TID photolabeling of -subunit) was fit to a single-site model f(x) = (100  A) / [1 + (x / IC50)] + A, with the IC50 value and A (the residual binding or subunit photolabeling) as adjustable parameters. TID inhibits [3H]tetracaine binding with an IC50 value of 2.4 ± 0.4 µM and a maximal inhibition of 95 ± 3%.

Mapping [¹²⁵I]TID Photoincorporation in nAChR M2 Segments. Suspensions of nAChR-rich membranes (5 mg, 2 mg/ml) were incubated with [¹²⁵I]TID in the absence of additional ligand and in the presence of 30 µM tetracaine, 50 µM PCP, or 30 µM HTX. The suspensions were photolyzed, and the nAChR -, -, and -subunits as well as the V8-20 fragment were isolated as described under Experimental Procedures. Typically, 100 to 200 µg of each subunit or of V8-20 were recovered. Based upon previous studies (Gallagher and Cohen, 1999), digestion of V8-20 or -subunit with EndoLys-C would produce fragments of ~10 kDa beginning at the N termini of the M2 segments that can be purified by Tricine SDS-PAGE and HPLC. For the -subunit, digestion with trypsin produces a similar fragment of ~7 kDa (White and Cohen, 1992). For nAChRs labeled with [¹²⁵I]TID in the absence or presence of amine NCAs, we also found that when aliquots of the subunit digests were fractionated by Tricine SDS-PAGE, for both V8-20 and the -subunit, the predominant [¹²⁵I]TID-labeled fragments had mobilities of ~10 kDa (Fig. 3). The presence of tetracaine or HTX, but not PCP, reduced ¹²⁵I incorporation in the - and -subunit fragments without the appearance of any novel labeled bands. Trypsin digestion of labeled -subunit produced a specifically labeled band of ~7 kDa (data not shown). The bulk of these digests was fractionated by Tricine SDS-PAGE on a preparative scale, and the material eluted from the gel bands was further purified by reversed phase HPLC (Fig. 4), from which the fractions containing the peak of ¹²⁵I were pooled for N-terminal sequence analysis.

View larger version (118K): [in this window] [in a new window]   Fig. 3.   Products of EndoLys-C digestion of [125I]TID-labeled nAChR subunits resolved by Tricine SDS-PAGE. V8-20 fragments (A) and -subunits (B) from nAChRs labeled with [125I]TID in the absence of additional ligand (lane 1), or in the presence of 50 µM PCP (lane 2), or 30 µM HTX (lane 3), or 30 µM tetracaine (lane 4) were digested with EndoLys-C as described under Experimental Procedures, and aliquots (8%) of each digestion mixture were run on an analytical Tricine SDS-PAGE gel. Shown here is an autoradiogram of the gel (1-week exposure) along with the migration distances of the prestained molecular mass markers.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Purification by reversed phase HPLC of [125I]TID-labeled fragments from proteolytic digests of nAChR subunits. nAChR-rich membranes were photolabeled with 6.5 µM [125I]TID in the absence of other NCAs () or in the presence of 30 µM tetracaine (+), 50 µM PCP (), or 30 µM HTX (). EndoLys-C digests of [125I]TID-labeled V8-20 and -subunits and trypsin digests of labeled -subunits were fractionated by Tricine SDS-PAGE. The bands of 8 to 10 kDa expected to contain fragments beginning at the N termini of M2 were excised and eluted as described under Experimental Procedures and then fractionated by reversed phase HPLC (Brownlee C-4 column, 0.2 × 10 cm) with the elution gradient increasing linearly from 25% to 100% solvent B (60% acetonitrile, 40% isopropanol, 0.05% trifluoroacetic acid) in 80 min. (Solvent A was 0.08% trifluoroacetic acid.) Flow was 0.2 ml/min, fractions were collected every 2.5 min, and total 125I cpm in each of the fractions was determined by gamma counting. Each of the profiles shown for a given subunit is from the same labeling experiment, but the data for the -subunit are from different labeling experiment than the data for the - and -subunits. There was no labeling experiment in which the effect of tetracaine on [125I]TID-labeling in -subunit was characterized at the same time as PCP and HTX, so for the -subunit, no data are included for tetracaine. For the -subunit, fractions 26 and 27 were pooled for sequence analysis, whereas for - and -subunits, fractions 28 to 31 and 25 to 26 were pooled. 125I cpm recovered in the pooled fractions: -subunit (A) (control, 63,900; +PCP, 40,000; +HTX, 13,300); -subunit (B) (control, 5,650; +tetracaine, 2,120; +PCP, 2,190; +HTX, 3,440); -subunit (C) (control, 26,090; +tetracaine, 4,540; +PCP, 33,600; +HTX, 7,500).

[¹²⁵I]TID Photoincorporation in the Presence of Tetracaine. Subunit fragments were isolated from nAChRs labeled with [¹²⁵I]TID in the absence and presence of 30 µM tetracaine. Sequence analysis of the purified -, -, and -subunit fragments each revealed a single sequence beginning at the N terminus of the M2 segments (Fig. 5). For each of the subunit fragments isolated from nAChRs that were labeled in the absence of tetracaine, there was ¹²⁵I release at cycles 9 and 13 of Edman degradation, corresponding to incorporation at Leu-251 (M2-9) and Val-255 (M2-13), Leu-257 (M2-9), and Leu-261 (M2-13), or Leu-265 (M2-9) and Leu-269 (M2-13). These results agree with the previous characterizations of amino acids in the nAChR M2 domain that are specifically labeled by [¹²⁵I]TID (Blanton and Cohen, 1992; White and Cohen, 1992). As was seen previously, within -subunit [¹²⁵I]TID was incorporated with greater efficiency at M2-9 than at M2-13, whereas for - and -subunits, the labeling at M2-13 was more prominent. Sequence analysis of the fragments isolated from nAChRs labeled in the presence of tetracaine similarly were characterized by ¹²⁵I release in the M2 segments only at positions 9 and 13 (Fig. 5), but for each subunit the efficiency of incorporation at M2-9 and M2-13 was reduced by 90% (Table 1A). When the data were combined from multiple labeling experiments, tetracaine at 30 µM reduced the efficiency of [¹²⁵I]TID labeling of M2-9, M2-13, M2-9, M2-13, M2-9, and M2-13 by 88 ± 4% (Table 2). There was no significant difference in the relative reduction of labeling in any of the residues (ANOVA, p = 0.76). Furthermore, there were no significant differences in the ratios of ¹²⁵I incorporation in M2-9 to M2-13 (p = 0.77) or M2-9 to M2-13 (p = 0.89) in the presence and absence of tetracaine. Thus, in the presence of tetracaine there was no evidence that [¹²⁵I]TID was shifted within the M2 ion channel domain to be in contact with amino acids other than M2-9 and M2-13, and the proportionate reduction of ¹²⁵I incorporation at all amino acids was consistent with mutually exclusive binding of either [¹²⁵I]TID or tetracaine.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Effect of tetracaine on [125I]TID incorporation within the M2 ion channel domain. A-C, 125I (, ) and picomoles of residues (, ) released during N-terminal sequencing of -, -, and -subunit fragments isolated by SDS-PAGE and reversed phase HPLC from nAChRs photoincorporated with [125I]TID in the absence (, ) and presence (, ) of 30 µM tetracaine. All data are from a single labeling experiment. For each subunit, the only sequence detected began at the amino terminus of the M2 segment, shown at the top of each panel. The initial (I0) and repetitive (R) yields of for each sample as well as the efficiency of incorporation into M2-9 and M2-13 are listed in the Table 1A. Insets, 125I release profiles for the samples labeled in the presence of tetracaine were plotted on an expanded scale to better visualize the pattern of 125I release.

View this table: [in this window] [in a new window]   TABLE 2 Effects of NCAs on [125I]TID incorporation in M2-9 and M2-13 of nAChR - and -subunits: averages from of multiple labeling experiments The efficiency of [125I]TID incorporation at M2-9 and M2-13 in the absence of additional ligand (control) or in the presence of tetracaine, PCP, or HTX was calculated as described under Experimental Procedures. From the data for each labeling experiment the ratios of labeling at M2-9 and M2-13 were calculated, and the averages of the ratios were determined for all experiments. In addition, for each position the percentage of reduction of labeling was determined from the efficiency of [125I]TID incorporation in the presence of NCA relative to control. The values tabulated are the averages of different labeling experiments ± the standard deviations. The number of labeling experiments used in the determinations is given in parentheses.

[¹²⁵I]TID Photoincorporation in the Presence of PCP and HTX. nAChR-rich membranes were incubated with [¹²⁵I]TID in the absence of additional ligand and in the presence of either 50 µM PCP or 30 µM HTX. These concentrations of NCAs were chosen to maximize binding to the high-affinity site while avoiding binding to the agonist site or to other low-affinity binding sites (Heidmann et al., 1983). At these concentrations, PCP and HTX each caused maximal change in of [¹²⁵I]TID incorporation into the nAChR as determined at the level of individual subunits (White and Cohen, 1988). The suspensions were photolyzed, and the nAChR -, -, and -subunits as well as the V8-20 fragment were isolated. HTX reduced ¹²⁵I incorporation in V8-20 by 80%, -subunit by 70%, -subunit by 80%, and -subunit by 50%. PCP reduced labeling in the -subunit by 20% and in the V8-20 fragment by 60%, but it increased labeling in the -subunit by 20% and in the -subunit by 10%. These results were generally consistent with previous studies (White and Cohen, 1988). Fragments of the -, -, and -subunits that begin at the N termini of the M2 segments were generated, purified, and sequenced (Figs. 6 and 7). For the fragments isolated from nAChRs labeled in the presence of PCP (Fig. 6) or HTX (Fig. 7) there were peaks of ¹²⁵I release in cycles 9 and 13, without the appearance of any novel labeled amino acids within the M2 segments. In contrast to tetracaine, PCP altered ¹²⁵I incorporation into each residue differently (Fig. 6; Table 1B). The addition of 50 µM PCP enhanced the labeling of M2-9 by 2-fold while reducing incorporation by 30 to 70% at the equivalent position in M2 and M2. In the presence of PCP there was no change in the labeling of M2-13. When the data were combined from multiple experiments (Table 2) these changes in the [¹²⁵I]TID labeling pattern were statistically significant by ANOVA (p = 0.007). Furthermore, the ratios of ¹²⁵I labeling in M2-9 to M2-13 were significantly different in the presence and absence of PCP in both M2 (p < 0.001) as well as M2 (p = 0.015).

View larger version (38K): [in this window] [in a new window]   Fig. 6.   Effect of PCP on [125I]TID incorporation within the M2 ion channel domain. A-C, 125I (, ) and picomoles of residues (, ) released during N-terminal sequencing of -, -, and -subunit fragments isolated by SDS-PAGE and reversed phase HPLC from nAChRs photoincorporated with [125I]TID in the absence (, ) and presence (, ) of 50 µM PCP. The data for the three subunits are from separate labeling experiments. For each subunit, the primary sequence detected began at the amino terminus of the M2 segment, and that sequence is shown at the top of each panel. No other sequences were detected at >10% of the primary sequence, except for the -subunit control, which contained a secondary sequence beginning at Lys-216 at the amino terminus of M1 at one-third the level of the primary sequence. The initial (I0) and repetitive (R) yields of for each sample as well as the efficiency of incorporation into M2-9 and M2-13 are listed in the Table 1B. Inset, 125I release () for the -subunit sample labeled in the presence of PCP plotted on an expanded scale.

View larger version (40K): [in this window] [in a new window]   Fig. 7.   Effect of HTX on [125I]TID incorporation within the M2 ion channel domain. A-C, 125I (, ) and picomoles of residues (, ) released during N-terminal sequencing of -, -, and -subunit fragments isolated by SDS-PAGE and reversed phase HPLC from nAChRs photoincorporated with [125I]TID in the absence (, ) and presence (, ) of 30 µM HTX. The controls for the - and -subunits are the same as in Fig. 6, because for those subunits the labelings with PCP and HTX were carried out in parallel. For each subunit, the primary sequence detected began at the amino terminus of the M2 segment, and that sequence is shown at the top of each panel. No other sequences were detected at >10% of the primary sequence, except for the -subunit control, which contained a secondary sequence beginning at Lys-216 at the amino terminus of M1 at one-third the level of the primary sequence. The initial (I0) and repetitive (R) yields of sequencing as well as the efficiency of incorporation into M2-9 and M2-13 are listed in Table 1C. Insets, 125I release () profiles for the samples labeled in the presence of PCP plotted on an expanded scale.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In these studies we examined the effects of tetracaine, PCP, and HTX on the pattern of photolabeling of [¹²⁵I]TID in the nAChR M2 ion channel domain. Previously [¹²⁵I]TID and [³H]tetracaine had each been shown by direct photolabeling to bind within the ion channel at the level of M2-9 and M2-13 in the closed state. Our results now demonstrate that tetracaine, the dimethylaminoethyl ester of p-butylaminobenzoic acid, which is charged at physiological pH, binds in a mutually exclusive manner with [¹²⁵I]TID, an uncharged benzene derivative. In contrast, for PCP, a three-ring aromatic tertiary amine, and HTX, a bulky two-ring secondary amine (Fig. 1), we found no evidence for simple, competitive interactions with [¹²⁵I]TID within the ion channel in the closed state. In the presence of PCP or HTX, there was no evidence that [¹²⁵I]TID was shifted to another locus within the ion channel domain, because [¹²⁵I]TID photolabeling was still restricted to amino acids M2-9 and M2-13. In the presence of HTX or especially PCP there was an alteration of the pattern of [¹²⁵I]TID labeling at M2-9 and M2-13 inconsistent with mutually exclusive binding. The simplest interpretation of the data is that in the absence of agonist, PCP, or HTX binds at another site, presumably in the ion channel domain, and the binding results in a subtle shift of the orientation of [¹²⁵I]TID bound at M2-9/13.

Tetracaine. In the analytical labeling experiments, high concentrations of tetracaine reduced [¹²⁵I]TID photoincorporation into the nAChR -subunit by 92 ± 1% (Fig. 1). This amount of inhibition would be expected for a drug that prevented [¹²⁵I]TID photolabeling within the M2 ion channel domain either by preventing the binding of [¹²⁵I]TID or by stabilizing the desensitized state of the nAChR (White and Cohen, 1988; 1992). However, tetracaine at concentrations up to 100 µM does not desensitize the nAChR (Boyd and Cohen, 1984; Middleton et al., 1999), and the IC₅₀ value of 3 µM for tetracaine inhibition is consistent with its binding to the M2 ion channel domain in the absence of agonist. In our studies, nonradioactive TID reduced the equilibrium binding of [³H]tetracaine by 95 ± 3%, a result consistent with competitive inhibition, although in view of the uncertainties, noncompetitive inhibition cannot be excluded.

PCP and HTX. In the absence of agonist PCP binding to its high-affinity site in the nAChR causes an allosteric inhibition of [¹²⁵I]TID photolabeling of the -, -, and -subunits and an increased photolabeling of the -subunit (White and Cohen, 1988; Ryan et al., 2001). HTX binding to its high-affinity site causes an allosteric inhibition of [¹²⁵I]TID photoincorporation into each nAChR subunit (White and Cohen, 1988). In our study, concentrations of PCP (50 µM) and HTX (30 µM) were used that in the previous studies were shown to cause maximal inhibition (or potentiation) of [¹²⁵I]TID photoincorporation into the nAChR as determined at the level of individual subunits. When we characterized by sequence analysis PCP's or HTX's effects on [¹²⁵I]TID incorporation in the M2 segments from nAChR -, -, and -subunits, we found that neither PCP nor HTX resulted in the labeling by [¹²⁵I]TID of any amino acids within M2 not labeled in the absence of the drugs. PCP (50 µM) enhanced the labeling of M2-9 by 2- to 3-fold and altered the efficiency of [¹²⁵I]TID incorporation into the other residues by significantly different percentages (Fig. 6; Table 1). Furthermore, the ratios of ¹²⁵I incorporation in M2-9 to M2-13 and M2-9 to M2-13 were significantly different from those observed in the absence of PCP (Table 2). This alteration in the pattern of labeling at M2-9/M2-13 suggests that PCP changes the orientation of [¹²⁵I]TID's diazirine with respect to the labeled residues, a result consistent with an allosteric interaction between PCP and TID. Similarly, the change in the ratio of [¹²⁵I]TID incorporation in M2-9 to M2-13 seen in the presence of HTX indicates that HTX also alters allosterically the structure of the TID binding domain.