Introduction

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The nicotinic acetylcholine receptor (AChR) from Torpedo californica electric organ is a pentameric, ligand-gated, nonspecific cation channel composed of four distinct, homologous subunits with a stoichiometry of ₂ (Raftery et al., 1980; Noda et al., 1983; Unwin, 1993). Each subunit possesses four putative transmembrane segments termed M1, M2, M3, and M4. Studies of chimeric AChRs (Imoto et al., 1986, 1988) have produced substantial evidence that the M2 domain lines the pore of the channel. Affinity-labeling experiments further identified this sequence as the site of high-affinity noncompetitive antagonist binding. Meproadifen mustard labeling was localized to Glu-262 in M2 (Pedersen et al., 1992); chlorpromazine and triphenylmethylphosphonium specifically label homologous residues within the M2 sequences of each subunit (Giraudat et al., 1986, 1987, 1989; Hucho et al., 1986). Labeling studies with TID, an uncharged noncompetitive antagonist, identified reactive sites within M2 for AChR stabilized in either the resting conformation or the desensitized state (White et al., 1991; White and Cohen, 1992). Mutagenesis of residues within the M2 sequence affects the affinity of open channel block by the high affinity noncompetitive antagonist (NCA) QX-222 (Leonard et al., 1988; Charnet et al., 1990). These data support a model for channel-blocking activity that is consistent with simple steric occlusion of the pore by NCAs (Neher and Steinbach, 1978).

The M2 locus is likely the high-affinity binding site for other noncompetitive antagonists such as phencyclidine (PCP), ethidium, and proadifen, whose binding loci have not yet been identified at the resolution of individual amino acids (Krodel et al., 1979; Heidmann et al., 1983; Herz et al., 1987). With some exceptions, noncompetitive antagonists are generally cationic and hydrophobic, but otherwise constitute a structurally heterogeneous class of ligands. The binding characteristics of several high-affinity NCAs have been studied extensively, resulting in a better understanding of receptor structure. NCAs such as tetracaine and TID preferentially bind the resting state of the AChR; histrionicotoxin shows a slight conformational preference, whereas most other NCAs, including phencyclidine, ethidium, meproadifen, chlorpromazine, triphenylmethylphosphonium, and quinacrine, preferentially bind the desensitized conformation of AChR at equilibrium (Krodel et al., 1979; Heidmann et al., 1983; White et al., 1991; Wu et al., 1994; Lurtz et al., 1997).

Fluorescent NCAs such as ethidium and quinacrine have been used to examine the structure and environment of the AChR. These compounds increase their quantum yield upon binding the NCA site. Fluorescence measurements have shown that ethidium becomes immobilized and shielded from solvent upon binding the AChR (Herz et al., 1987; Herz and Atherton, 1992). Quenching and fluorescence energy transfer measurements are potentially useful tools for further characterization of the properties of the NCA site. The utility of these two fluorophores is somewhat limited by their affinity, the sensitivity of their affinities to ionic strength changes (Lurtz et al., 1997), and nonspecific interaction with the lipid bilayer. We desired to carry out diffusion-enhanced energy transfer experiments (Stryer et al., 1982) using NCA site ligands as the energy transfer acceptors. The sensitivity of such experiments is strongly dependent on the extinction coefficient of the ligand. We therefore investigated the ability of strongly absorbing dyes to act as NCAs of the AChR.

We present data that the aminotriarylmethane dyes, compounds with structures similar to the NCA, triphenylmethylphosphonium, are a novel family of NCAs. Several members of this family bind with K_D values in the low nanomolar range, particularly crystal violet (CrV). Further analysis of CrV demonstrated that crystal violet binds the noncompetitive antagonist site in the pore of the AChR with high affinity and a substantial, concomitant fluorescence enhancement. Its high affinity and fluorescence properties further makes CrV, in particular, a good probe for future fluorescence quenching and fluorescence energy transfer experiments. The high affinity of this compound and the existence of many structurally related basic dyes will provide an approach to a more detailed structure-activity relationship of the NCA site. Here we present an analysis of the binding relationships of these ligands.

	Experimental Procedures

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Materials. Crystal violet hydrochloride, methyl violet 2B, brilliant green, malachite green carbinol hydrochloride, ethyl violet hydrochloride, methyl green zinc chloride, rosaniline, pararosaniline chloride, new fuchsin, patent blue VF, Victoria pure blue BO, leuco crystal violet, and crystal violet lactone were obtained from Aldrich Chemical Co. (Milwaukee, WI). N-(4-(((4-dimethylamino)phenyl)(4-methoxy-3-sulfophenyl)methylene)-2,5-cyclohexadien-1-ylidene)-N-methylmethanaminium inner salt (Dpmsm) was obtained from Eastman Chemical Co. (Rochester, NY). Before use, crystal violet was crystallized from chloroform, then recrystallized from water. Purity was assessed by reversed-phase high performance liquid chromatography (HPLC) as described below.

Spectroscopy. Fluorescence measurements were taken on either an SLM 8000C fluorometer (Rochester, NY) with a 350W xenon arc lamp or on an ISS PC1 fluorometer (Urbana, IL) equipped with a 300W lamp using 10 × 10 mm cuvettes. For single-point measurements or time-dependent measurements of CrV fluorescence, the excitation wavelength was 600 nm with a 550-nm cut-on filter (Oriel no. 59502; Stratford, CT); emission was monitored at 645 nm with an RG630 cut-on filter (ESCO no. 5274630); all bandwidths were 16 nm. Fluorescence excitation spectra were collected as a ratio of fluorescence signal intensity to a reference signal to correct for lamp intensity at the various wavelengths. Emission spectra were collected on the ISS instrument; they were corrected for instrument response using calibration factors provided by the manufacturer. Excitation and emission spectra were also corrected for inner filter effects using the following equation (Lakowicz, 1983): F_corr = F_obs · 10^(Aex+Aem)/2, where F_obs is the observed fluorescence intensity, A_ex and A_em are the absorbances of the fluorophore at the excitation and emission wavelengths, and F_corr is the fluorescence intensity corrected for inner filter effects. This correction was only significant for spectra taken at high CrV concentrations.

Binding Assays. [³H]PCP inhibition assays were conducted by centrifugation assay essentially as described (Lurtz et al., 1997), except that some experiments were incubated for 2 h at ambient temperature. Data were fit to a model for inhibition at a single site: B = B_max/(1 + I/K_app) + Bcg (eq. 1), where B_max represents the maximal binding, I is the inhibitor concentration, K_app is the inhibition constant, and Bcg is the background (nonspecific) binding, as defined by the presence of excess unlabeled competitor. [³H]ACh-binding assays were conducted as described previously (Pedersen and Papineni, 1995); nonspecific binding was determined by inclusion of excess carbamylcholine.

²²Na Efflux Assay ²²Na efflux assays were conducted using the procedure of Neubig and Cohen (1980) essentially as described by White et al. (1991); all manipulations were carried out at 4°C. Briefly, a concentrated suspension of AChR-rich vesicles was incubated with ²²Na overnight in HTPS. The excess ²²Na was removed by passing the vesicles over a 3-ml Dowex 50W-X8 column, and the vesicles were diluted immediately with HTPS. After a 20-min incubation to permit passive release of ²²Na to reach a slow, steady-state level, the assays were initiated by addition of 0.3 mM phenyltrimethylammonium and the suspension was filtered through Whatman GF/F filters 20 s later. The filters had been pretreated with 1% polyethyleneimine and washed with 10 ml HTPS before filtration. To measure block of efflux by NCAs, the vesicles were preincubated for 10 min with the NCA before initiating release. Maximal ²²Na efflux from Torpedo californica vesicles was determined by 10-min incubation with gramicidin D. Data were normalized to the release observed in the presence of gramicidin.

Determination of Partition Coefficients. AChR-rich membranes were incubated for 1 h at ambient temperature in the presence of 500 nM CrV and 0.1 mM carbamylcholine in the presence or absence of 1 mM PCP. Membranes were sedimented in a tabletop TOMY MTX-150 microcentrifuge at 18,500g for 30 min at 20°C, and supernatants then were transferred to microfuge tubes containing CH₃CN (50-55%) with 0.1% TFA and assayed by HPLC. Crystal violet was stable in 0.1% TFA for several days. To release bound CrV, the sedimented membranes were resuspended in 55% CH₃CN/0.1% TFA plus 1 mM PCP. The membranes were removed by centrifugation, as above, and the crystal violet released into the supernatant then was assayed by HPLC. HPLC was carried out on a Beckman 125 System Gold, with a model 166 variable-wavelength detector (254 nm used routinely), and data collection was carried out with System Gold software. The separation was on a C18 Ultrasphere column (250 × 4.6 mm; Beckman Instruments) at 1 ml/min using a gradient of CH₃CN in 0.1% TFA to elute CrV. CrV concentrations were determined by comparison of the HPLC peak area to a standard curve. Injecting various known amounts of CrV generated standards.

1

	Results

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Aminotriarylmethane dyes (Fig. 1) share structural similarities with many known NCAs of the AChR. Such similarities include the presence of aromatic groups, tertiary or quaternary amines, and a positive charge. Although this family of basic dyes normally is nonfluorescent, they have high extinction coefficients with absorption maxima in the visible part of the spectrum. To evaluate whether some of the aminotriarylmethane dyes would bind as noncompetitive antagonists of the AChR, we initially screened several of these dyes for their affinity for the NCA site by competitive radioligand-binding assays.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Structures of aminotriarylmethane dyes. The figure shows the structure of crystal violet and other dyes examined in this study.

Aminotriarylmethane Dyes Inhibit [³H]Phencyclidine Binding. The ability of several aminotriarylmethane dyes to inhibit [³H]PCP binding to AChR from Torpedo californica electric organ was assessed by a centrifugation assay in Torpedo physiological saline (HTPS) using low [³H]PCP concentrations (~1 nM). Preliminary experiments had indicated that addition of the agonist carbamylcholine yielded lower K_app values for several of the dyes. Therefore, 100 µM carbamylcholine was routinely included when assaying dyes for binding. Sample curves for five of the dyes are shown in Fig. 2. Each set of data fit well to a curve for inhibition of a single site. The K_app values determined from the nonlinear regression (solid lines, Fig. 2) range from 50 nM for crystal violet to 360 µM for Dpmsm, a zwitterionic analog of crystal violet. Table 1 summarizes the measured inhibition constants for all of the compounds tested. Crystal violet, ethyl violet, and methyl violet 2B had K_app values of less than 100 nM, values that are substantially lower than those for previously characterized NCAs. New fuchsin, brilliant green, and Victoria blue BO had K_app values comparable to those of other high-affinity NCAs in the range of 100 nM to 1 µM. The remaining compounds had K_app values in the micromolar range, comparable to many other NCAs. Dpmsm had an exceptionally high K_app that likely reflects the presence of a negative charge in the structure.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Inhibition of [3H]phencyclidine binding by aminotriarylmethane dyes. AChR-rich membranes (100 µg; 40 nM AChR, 1 ml volume) were incubated in HTPS buffer at ambient temperature (20°C) with 1 nM [3H]PCP in the presence of 100 µM carbamylcholine and the indicated concentrations of dye. Bound [3H]PCP was determined as described under Experimental Procedures. Data from each curve were fit to eq. 1 (solid lines), and Kapp values were determined for each: Dpmsm, 360 µM (), pararosaniline, 1.6 µM (), brilliant green, 340 nM (), new fuchsin, 190 nM (), and crystal violet, 28 nM (). Each symbol represents the average of duplicate determinations. Data from separate experiments were normalized to the maximum [3H]PCP bound and combined in a single figure.

Fluorescence and Absorbance Spectra of Crystal Violet. CrV is essentially nonfluorescent in aqueous solution (Fig. 3A) but had been reported to acquire fluorescence upon binding protein (Anderson et al., 1996; Baptista and Indig, 1998). Therefore, we examined the fluorescence of three higher-affinity dyes in the presence of the AChR: CrV, ethyl violet, and new fuchsin. Preliminary experiments indicated that CrV had substantial specific fluorescence enhancement upon binding the AChR. The other two compounds also exhibited fluorescence when added to AChR-rich membranes, but their fluorescence was less susceptible to inhibition by PCP. This suggested less specific fluorescence upon binding the NCA site or substantial fluorescence due to nonspecific interactions.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Fluorescence and absorbance spectra of crystal violet. A, excitation spectra (traces 1 and 2) and emission spectra (traces 4 and 5) for 50 nM crystal violet incubated in HTPS with 100 nM AChR and 1 mM carbamylcholine were collected as described in Experimental Procedures. Spectra were taken in the absence (spectra 1 and 4) or presence of 100 µM PCP (spectra 2 and 5). Excitation and emission spectra of 10 µM crystal violet in HTPS without AChR present are shown for comparison (spectra 3 and 6, respectively). Each set of excitation and emission spectra was normalized to the peak value for CrV bound to the AChR. B, absorbance spectra of 400 nM crystal violet in HTPS (spectrum 1) or bound to AChR-rich vesicles (512 nM AChR, spectrum 2). The inset shows the structure of crystal violet chloride. The peak wavelengths for the spectra are as follows: A, spectra 1, 599 nm; 2, 599 nm; 3, 593 nm; 4, 626 nm; 5, 633 nm; 6, 640 nm. B, spectra 1, 591 nm; 2, 600 nm.

Interaction of Crystal Violet with Membranes. Although the majority of the fluorescence enhancement observed upon CrV binding the AChR was inhibitable by PCP, there was substantial residual fluorescence likely due to nonspecific interactions with the lipid bilayer or with other proteins. Therefore, we used HPLC to quantitate nonspecific interaction with AChR-rich vesicles; this was carried out in high concentrations of blocking agents to prevent specific interaction with the high-affinity NCA site on the AChR (see Experimental Procedures). We measured the partition coefficient of CrV in both physiological buffer (HTPS, high ionic strength) and at low ionic strength (20 mM HEPES). CrV has a high partition coefficient of 2.62 ± 0.26 (mg/ml)¹ (n = 4) in physiological buffer. Compare these values with the much lower values determined for ethidium, PCP, and quinacrine (Lurtz et al., 1997) that range from 0.05 to 0.17 (mg/ml)¹. In low-ionic-strength buffer, the partition coefficient is 7.48 ± 0.86 (mg/ml)¹, (n = 4), a value similar to quinacrine, but somewhat higher than ethidium [2 (mg/ml)¹] or PCP [0.11 (mg/ml)¹]. Thus, under typical conditions used for measuring binding and fluorescence to the AChR (~0.1 mg/ml AChR-rich vesicles), we expect a significant amount (~20%) of nonspecific interaction with the vesicles.

Crystal Violet Binding Measured by Fluorescence Enhancement. The relatively high concentration of AChR (40 nM) limited the ability to measure true K_D values less than this value by inhibition of [³H]PCP binding. This concentration was near the K_app values observed for CrV, methyl violet, and perhaps ethyl violet. In these cases, the K_app value may reflect titration of the binding sites rather than the true K_D. Therefore, we took advantage of the fluorescence of CrV to measure its K_D for interaction with the AChR by fluorescence enhancement in varying concentrations of CrV (Fig. 4).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Equilibrium-binding crystal violet as measured by fluorescence intensity. AChR-rich membranes (20 nM AChR, 1.5 ml) were incubated in HTPS at ambient temperature with varying concentrations of crystal violet in the absence () or presence () of 0.1 mM PCP. The experiments were either in the presence (A) or in the absence (B) of 100 µM carbamylcholine. The fluorescence intensity was measured for each sample as described in Experimental Procedures. After removal of bound crystal violet by centrifugation, free crystal violet was quantified by fluorescence in CHAPS and as described in Experimental Procedures. Specific binding () was determined after subtraction of nonspecific binding from the fluorescence intensity measured in the absence of PCP. The solid lines represent the best fits to the binding equation (eq. 2). As determined by the fitting, the KD was 7.6 nM and the maximal specific fluorescence was 0.096 (A), and the KD was 93 nM and the maximal specific fluorescence was 0.087 (B).

Crystal Violet Interaction with the Agonist-Binding Sites. The approximately 10-fold decrease in the K_D value for CrV in the presence of agonist suggested that a reciprocal effect of CrV on agonist binding should exist. We examined the affinity of [³H]ACh in varying concentrations of CrV (Fig. 5). Using low concentrations of AChR and low concentrations of high specific radioactivity [³H]ACh, we measured the ratio of bound to free [³H]ACh at increasing concentrations of CrV or proadifen (Fig. 5). At conditions in which only a small percentage of the sites are bound with ligand, the bound-to-free ratio of [³H]ACh is proportional to the affinity of [³H]ACh (i.e., inversely proportional to the K_D). The bound-to-free ratio increases nearly 10-fold in the presence of either added ligand, consistent with an increase in [³H]ACh affinity to the AChR. The CrV curve is to the left of the proadifen curve, indicating that CrV was more potent, consistent with its higher affinity. The decrease in the bound-to-free ratio of [³H]ACh observed at the higher concentrations of NCA likely reflects direct competition for binding at the agonist sites. CrV appears to interact with the ACh sites with an IC₅₀ of 30 µM and with proadifen with an IC₅₀ of ~300 µM.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Crystal violet increases [3H]ACh affinity. AChR-rich membranes (12 nM AChR) were incubated in HTPS in the presence of ~1 nM [3H]ACh and in the presence of increasing concentrations of either proadifen () or CrV (). Bound and free concentrations of [3H]ACh were determined as described in Experimental Procedures. Data were plotted as bound divided by free [3H]ACh. Each curve illustrates the increase in [3H]ACh binding due to an increased affinity for the AChR in the presence of desensitizing ligand.

Crystal Violet Binds Competitively with the PCP. Fluorescence binding data (Fig. 4) and [³H]PCP inhibition data (Fig. 2) were consistent with CrV binding to the high-affinity noncompetitive antagonist site of the AChR. On this basis alone, however, an allosteric mechanism of inhibition could not be ruled out altogether. Other NCAs have multiple sites of binding associated with the AChR (Heidmann et al., 1983) and may be able to influence the conformation of the AChR through nonspecific interactions (Boyd and Cohen, 1984). We therefore examined the PCP inhibition of CrV binding at several CrV concentrations, using CrV fluorescence as an indicator of binding. For each curve, the CrV concentration was held constant and the fluorescence was measured in the presence of increasing PCP concentrations (Fig. 6).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Inhibition of crystal violet fluorescence by phencyclidine. AChR-rich membranes (20 nM) were incubated in HTPS in the presence of 0.1 mM carbamylcholine, with 25 nM (), 50 nM (), 100 nM (), 200 nM (), or 400 nM () crystal violet and increasing concentrations of PCP. The fluorescence intensity at each PCP concentration for each crystal violet concentration is plotted. Symbols represent the average of duplicate determinations. Each curve was fit to a binding isotherm for a single site inhibition. Kapp values for PCP were replotted as a function of CrV concentration (inset). The slope of the replot was determined by linear regression.

Stoichiometry of Crystal Violet Binding to the AChR. Binding data from both radioligand inhibition assays and direct binding experiments are consistent with CrV binding to a single class of sites on the AChR. To quantitate the number of CrV-binding sites on the AChR associated with the fluorescence enhancement, CrV was titrated into a concentrated suspension of AChR-rich vesicles and the fluorescence was measured (Fig. 7). At low concentrations, nearly all of the CrV added bound to the AChR and the fluorescence increased linearly; the slope of this line reflects the fluorescence yield (per nM CrV) due to binding the AChR. At higher concentrations of CrV, the binding sites become saturated, and the fluorescence signal increased linearly with a lower slope. A similar slope was observed at the higher CrV concentrations titrated into AChR-rich vesicles in the presence of PCP to block binding to the NCA site. The limiting slope at the higher CrV concentrations, therefore, reflects the fluorescence increase due to nonspecific interactions. At the intersection of these lines, the added CrV concentration equals the total binding-site concentration (see Experimental Procedures). This value can be compared with the AChR concentration added based on [³H]ACh binding, which routinely is used to quantitate various AChR-rich vesicle preparations. The ratio is 1.06 ± 0.17 CrV binding sites per AChR for four independent determinations carried out as shown in Fig. 7. This value suggests that CrV binds a single high-affinity site on the AChR, which is consistent with binding to the central ion pore.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Stoichiometry of crystal violet binding to the AChR. AChR-rich membranes (375 nM AChR) were incubated in the presence of 0.1 mM carbamylcholine in HTPS (). CrV was titrated into the membranes, and the fluorescence intensity was monitored. The titration was repeated in the presence of 1 mM PCP () to define nonspecific fluorescence. Linear regressions were applied to initial and ending linear components of the titration in the absence of PCP (0-200 nM CrV and 650-1000 nM CrV, respectively). The intercept from these two sets of points is 376 nM and defines the concentration of CrV-binding sites (see Experimental Procedures).

Crystal Violet Blocks ²²Na Efflux from AChR-Rich Vesicles. To determine the effect of CrV on AChR function, we examined the ability of CrV to block ²²Na efflux from AChR-rich vesicles. Efflux was elicited by the addition of the partial agonist phenyltrimethylammonium (PTMA). Preliminary experiments established that 0.3 mM PTMA yielded maximum agonist-stimulated efflux, a level of efflux that was usually 60 to 70% of that released by the ionophore gramicidin. Preincubation of AChR-rich vesicles with a CrV concentration of 1 to 10 µM blocked agonist-stimulated efflux with an average IC₅₀ of 1.7 ± 0.8 µM (Fig. 8; Table 2). This value can be compared to the average IC₅₀ of 19 ± 6 µM for PCP. This IC₅₀ was consistent with that observed previously by others (White et al., 1991).

View larger version (26K): [in this window] [in a new window]   Fig. 8.   Crystal violet inhibits 22Na efflux from Torpedo californica vesicles. AChR-rich vesicles (90 nM AChR) that had been loaded with 22Na were incubated in HTPS (1 ml) in the presence of varying concentrations of CrV () or PCP () for 10 min on ice. 22Na efflux was initiated by the addition of PTMA (0.3 mM final concentration). After 20 s, samples were immediately filtered and washed with 10 ml ice-cold HTPS as described in Experimental Procedures. Data are plotted as the percentage of counts released relative to the release induced by gramicidin. Gramicidin-releasable counts typically ranged from 400 to 800 cpm with a filter background of 200 to 400 cpm. The IC50 for inhibition of efflux by CrV was 1.3 µM and by PCP was 25 µM. Each symbol and error bar represent the average and S.D. of three determinations.

	Discussion

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We have shown that various basic aminotriarylmethane dyes bind to the NCA site of the AChR. By characterizing one of these dyes, CrV, in detail we have shown that it possesses the characteristics of many high-affinity noncompetitive antagonists that bind the M2 region of the AChR and block activity by steric hindrance of ion passage through the pore of the AChR. These characteristics include high-affinity binding (~10 nM) for a single site on the AChR, higher affinity in the presence of agonist than for the resting conformation, competitive binding with PCP, the ability to allosterically interact with the agonist-binding sites, and the ability to block ²²Na efflux from AChR-rich vesicles. The K_app values for these properties are summarized in Table 2. There is good agreement between the potencies of CrV to inhibit ²²Na efflux, enhance [³H]ACh affinity, and block [³H]PCP binding in the absence of agonist (data not shown), all at concentrations near 1 µM. The primary difference is between those values and the directly measured K_D in the absence of agonist (100 nM, Table 2). The reason for this difference is not entirely clear, but may reflect difficulty in obtaining precise data for the K_D by fluorescence enhancement because of the higher nonspecific binding at the CrV concentrations required to measure binding in the absence of agonist.

The discrepancy between the K_app measured by fluorescence enhancement and by [³H]PCP binding in the presence of agonist (Table 2) simply reflects the lower limit of the [³H]PCP-binding assay; the value determined by fluorescence likely reflects the true K_D. Comparison of the K_app values in the absence of agonist versus those in the presence of agonist suggests that CrV binding strongly favors the desensitized conformation of the AChR because this is the conformation stabilized by agonists at equilibrium. Nonetheless, CrV potentially could be acting as an open channel blocker or trapping the AChR in a distinct conformation with high affinity for agonist. These possibilities may be resolved by examination of the kinetics of binding using either fluorescence methods or by single channel measurements of blockade. The preferential binding in the presence of agonist is consistent with the observation that larger NCAs preferentially bind the desensitized state of the AChR, whereas ligands that prefer binding to the resting conformation, such as tetracaine or TID, tend to be smaller (Cohen et al., 1986; White and Cohen, 1992). This trend also favors the model of Furois-Corbin and Pullman and of White and Cohen that suggests that the pore of the AChR expands at the synaptic end upon desensitization by tilting of the M2 -helices away from the central axis of the AChR (Furois-Corbin and Pullman, 1989; White and Cohen, 1992).

Several of the other dyes bind with affinities comparable to CrV, and many of the dyes display similar properties, including higher affinity for the desensitized state as measured by inhibition of [³H]PCP binding (brilliant green, malachite green, rosaniline, ethyl violet; data not shown). Therefore, the affinities of the various dyes can be compared in terms of binding a single site on the AChR. The detailed structure-activity relationship of these dyes will be particularly interesting as they represent a large class of compounds with well developed chemistry. This will permit a closer examination of the structure of the pore of the AChR than has been possible with the previously known heterogeneous collection of ligands.

Structure Activity Relationship of the Basic Dyes. The dyes examined have several discrete, small changes in structure that cause significant changes in affinity (see Fig. 1 for reference). The six methyls on the CrV-amines contribute substantially to affinity. Pararosaniline has none of these methyls and binds with ~200-fold lower affinity (compare K_app = 2200 nM for pararosaniline, Table 1, with K_D = 10 nM for CrV, Table 2). Removal of one complete dimethylamino group also substantially lowers affinity (compare malachite green, K_app = 3.9 µM, with CrV, K_D = 10 nM). Increasing the size of the amine substituents from methyl to ethyl groups improves affinity: compare the K_app values of malachite green and brilliant green. Likewise, ethyl violet has a K_app similar to CrV, but because the K_app estimate was limited by the AChR concentration, a precise comparison will require further experiments.

Fluorescence of Crystal Violet. The fluorescence enhancement that accompanies binding of CrV to the AChR is greater than 200-fold and is due to a change in quantum yield, as shown by the small changes in the absorbance spectrum (Fig. 3). This large enhancement is possible because CrV is only weakly fluorescent in aqueous solution. The weak fluorescence is attributed to rapid de-excitation of the excited state by concerted rotation of the three aryl rings (Duxbury, 1993). Binding to the AChR likely inhibits this rotation, eliminating this mechanism of de-excitation, and results in a much higher quantum yield. The Stokes shift, the difference between the peak emission and absorbance wavelengths, decreased substantially upon AChR binding, indicative of binding in a nonpolar environment (Lakowicz, 1983). This conclusion is consistent with the generally nonpolar nature of the residues associated with the M2 sequences of the AChR that comprise the high-affinity NCA-binding site.