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J. Biol. Chem., Vol. 275, Issue 29, 22452-22460, July 21, 2000

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Biotinylation of Substituted Cysteines in the Nicotinic Acetylcholine Receptor Reveals Distinct Binding Modes for -Bungarotoxin and Erabutoxin a^*

Armin Spura^§, Ryan U. Riel, Neal D. Freedman, Shantanu Agrawal, Christopher Seto^¶, and Edward Hawrot

From the  Department of Molecular Pharmacology, Physiology, and Biotechnology, Division of Biology and Medicine, and the ^¶ Department of Chemistry, Brown University, Providence, Rhode Island 02912

Received for publication, February 15, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although previous results indicate that -subunit residues Trp¹⁸⁷, Val¹⁸⁸, Phe¹⁸⁹, Tyr¹⁹⁰, and Pro¹⁹⁴ of the mouse nicotinic acetylcholine receptor are solvent-accessible and are in a position to contribute to the -bungarotoxin (-Bgtx) binding site (Spura, A., Russin, T. S., Freedman, N. D., Grant, M., McLaughlin, J. T., and Hawrot, E. (1999) Biochemistry 38, 4912-4921), little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at 184-197 with the thiol-specific biotin derivative (+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine, we now show that only very subtle differences in reactivity (~10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin -Bgtx is significantly retarded for positions W187C, F189C, and reduced wild-type receptors (Cys¹⁹² and Cys¹⁹³), further emphasizing their major contribution to the -Bgtx binding site. Interestingly, although biotinylation of position V188C is not affected by the presence of -Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position V188C, but not at W187C or F189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The nicotinic acetylcholine receptor (AChR)¹ is the major prototype for neurotransmitter-gated ion channels and is found at high concentrations in the postsynaptic membranes of muscle cells, where it mediates the rapid propagation of electrical signals at the neuromuscular junction. It is a pentameric protein composed of four subunit types in a molar ratio 2::: (see Ref. 1 for review).

An important first step in assessing the structure and function of such ion channels at a molecular level is to determine their transmembrane topology. To address this issue, a variety of techniques have been developed (see Ref. 2 for review). The most commonly used methods are the epitope protection assay (3-5), in which an epitope that is recognized by a specific antibody is fused to the protein of interest, and N-linked glycosylation tagging, wherein N-linked glycosylation sites can be engineered into the protein under investigation and glycosylation can then be evaluated (6-8).

The above methods, however, are only useful to assess overall topology of membrane proteins. For the nAChR, there is general consensus on the overall topology, although final proof will have to await high resolution structural data; each subunit possesses a large, extracellular amino-terminal domain, which is followed by four transmembrane-spanning regions and a short extracellular carboxyl terminus (9). In contrast, the key structural issue of whether individual residues are solvent-exposed has not been resolved. To address this issue, Gallivan et al. (10) have recently employed the in vivo nonsense suppression technique to incorporate derivatives of the unnatural amino acid biocytin into the nAChR heterologously expressed in Xenopus oocytes. By evaluating the binding of ¹²⁵I-streptavidin to biotinylated receptors, they studied the surface exposure of individual residues comprising the main immunogenic region (spanning positions 67-76; Ref. 11) and showed that position 70 was highly exposed.

In the current study, we have used the substituted cysteine accessibility method (12, 13) to systematically map the accessibility of individual residues between positions 184 and 197 of the -subunit, the main determinant for agonist and competitive antagonist binding to the nAChR (14-16). To achieve this, we have introduced cysteine residues into the nAChR and labeled them with thiol-reactive, water-soluble biotin derivatives. Subsequently, we precipitated biotinylated receptors with immobilized streptavidin and probed the immunoprecipitates by Western blotting. Previous studies of oocyte-expressed Cys substitution mutations of -subunit residues 181-197 (17) indicate that the majority of these substitutions are well tolerated and lead to minimal perturbations in receptor function.

Here, we show that positions 184-197 are all amenable to biotinylation, suggesting that the entire region is surface-exposed. In addition, modifications with the uncharged biotin derivative occur with similar rates for all of these residues. Moreover, we demonstrate that preincubation with the competitive antagonist -Bgtx, a long-chain -neurotoxin, selectively blocks biotinylation of positions 187, 189, and cysteines 192 and 193 in reduced wild-type receptors, demonstrating the importance of these residues in the binding of -Bgtx and further supporting results we obtained previously (16). In contrast, preincubation with Ea, a short-chain -neurotoxin, yields a different footprint, preventing only position 188 and reduced wild-type receptors from biotinylation. Thus, our findings strongly argue that long- and short-chain -neurotoxins interact selectively with different positions when blocking agonist binding on the nAChR.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Reagents

MTSEA-biotin was from Toronto Research. PEO-biotin and streptavidin-agarose beads were from Pierce, mAb 35 from Research Biochemicals International, and Protein G-agarose beads from Santa Cruz Biotechnology.

Mutagenesis

We used a cytomegalovirus-based expression vector (GWI, British Biotechnology, Oxford, United Kingdom) to express the cDNAs for the -, -, -, and -subunits of the mouse muscle nicotinic AChR. Mutations were introduced using the Quikchange mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's specifications. Mutations were confirmed by diagnostic restriction enzyme digests and bidirectional sequencing of the entire insert following a DyeDeoxy terminator protocol (Perkin-Elmer).

Transfections and Cell Lines

These have been described previously (16).

Synthesis of N-Methanethiolsulfonyl-N'-biotinyl-2,2'-(ethylenedioxy)bis(ethylamine) (MTSEDE-biotin)

MTSEDE-biotin belongs to a class of compound generally known as alkyl alkanethiolsulfonates. It was synthesized as follows (Fig. 1).

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Synthesis scheme for MTSEDE-biotin.

Step 1: N-Boc-2,2'-(ethylenedioxy)bis(ethylamine) Derivative (18)-- 2,2'-(Ethylenedioxy)bis(ethylamine) (38 g, 257 mmol) was dissolved in 40 ml of deionized water and 40 ml of dioxane. While this solution was stirring, di-tert-butyl dicarbonate (8 g, 36.7 mmol) dissolved in 80 ml of dioxane was added dropwise. Formation of the bis-Boc-protected product was discouraged by performing the dropwise addition at 0 °C. The reaction was warmed to room temperature and stirred for 6 h. The solvents were removed by rotary evaporation, and the product was dissolved in ethyl acetate and washed with saturated NaHCO₃. The organic layers were dried over sodium carbonate, gravity-filtered, and rotary-evaporated to remove ethyl acetate. Flash chromatography using a 20% MeOH, 1% NH₄OH, CH₂Cl₂ solvent system gave a 66% yield (6 g, 2.4 mmol) of a yellowish oil.

Step 2: N-Boc-N'-biotinyl-2,2'-(ethylenedioxy)bis(ethylamine) Derivative (18)-- The mono-Boc-protected diamine (0.38 g, 0.15 mmol) was dissolved in 8 ml of methanol. To this solution was added diisopropylethylamine (1.2 g, 0.9 mmol) and biotin N-hydroxysuccinimide ester (0.78 g, 0.2 mmol). The flask was stirred at room temperature for 4 h, concentrated by rotary evaporation, and redissolved in 50 ml of ethyl acetate. This solution was washed once with 10 ml HCl, once with 10 ml of NaHCO₃, and once with brine. The organic layer was dried over magnesium sulfate, gravity-filtered through celite, and rotary-evaporated. Flash chromatography with an 8% MeOH/CH₂Cl₂ solvent system gives a 69% yield (0.5 g, 0.1 mmol) of a white solid.

Step 3: N'-Biotinyl-2,2'-(ethylenedioxy)bis(ethylamine) Derivative (19)-- The Boc-protected biotin diamine (0.5 g, 0.1 mmol) was dissolved in 4.9 ml of trifluoroacetic acid and stirred at room temperature for 20 min. The reaction flask was rotary-evaporated to remove trifluoroacetic acid, producing a brownish oil. This oil was dissolved in a few drops of deionized water and placed under vacuum to remove any remaining trifluoroacetic acid. Thin layer chromatography (15% MeOH/CH₂Cl₂) showed the product spot very close to the base line, suggesting that it contained the extremely polar free amine.

Step 4: N-Iodoacetyl-N'-biotinyl-2,2'-(ethylenedioxy)bis(ethylamine) Product-- The biotin diamine (0.06 g, 0.02 mmol) was dissolved in 2 ml of tetrahydrofuran and stirred at room temperature. To this solution, iodoacetic anhydride (0.17 g, 0.05 mmol) and diisopropylethylamine (0.12 g, 0.1 mmol) were added. The reaction was allowed to proceed for 1 h, after which the flask was rotary evaporated to remove the tetrahydrofuran and placed under vacuum. Flash chromatography was performed using 10% MeOH/CH₂Cl₂ as the eluent, giving an 88% yield of an off-white solid (0.06 g, 0.01 mmol).

Step 5: N-Methanethiolsulfonyl-N'-biotinyl-2,2'-(ethylenedioxy)bis(ethylamine)-- The iodobiotin diamine (0.75 g, 1.4 mmol) was taken up in 10 ml of dimethylformamide (DMF). To this solution was added sodium methanethiolsulfonate (0.37 g, 0.003 mmol), and the reaction was allowed to stir at room temperature for 2 h. The flask was placed under high vacuum to remove the DMF, and the product was flash-chromatographed using a 15% MeOH/CH₂Cl₂ solvent system. An 88% yield (0.64 g) of a yellowish oil was isolated. NMR spectra for MTSEDE-biotin and its intermediates were determined to confirm the purity of the product (data not shown) and are available upon request.

Step 6: Biotin N-Hydroxysuccinimide Ester (20)-- Biotin (5.5 g, 22.6 mmol) was dissolved in 70 ml of DMF. To this solution was added N-hydroxysuccinimide (3.12 g, 27.1 mmol) and diisopropyl carbodiimide (3.42 g, 27 mmol). The reaction was stirred at 90 °C overnight, after which rotary evaporation was used to remove DMF, giving a yellowish solid. Ethyl ether (150 ml) was added to the crude product to dissolve impurities, after which the crude solid was suction filtered. This off-white solid had a melting point range of 177-182 °C. The crude product was recrystallized in isopropanol and suction-filtered to give an 84.5% yield of a white solid (6.5 g, 19 mmol). The melting point of this solid was 200-202 °C.

Step 7: Sodium Methanethiolsulfonate (21)-- Sodium hydrosulfide was dried over P₂O₅ for 3 days. This dried sodium hydrosulfide (11 g, 20 mmol) was then dissolved in 150 ml of absolute ethanol. Methanesulfonyl chloride (11.4 g, 9.9 mmol) was added dropwise to this solution as it stirred at room temperature and under a nitrogen atmosphere. After all of the methanesulfonyl chloride had been added, the reaction was allowed to stir for another 2 h, under nitrogen. As nitrogen gas was bubbled through the reaction, it was forced through a drying tube and then bubbled through 2000 ml of bleach in order to neutralize the developing hydrogen sulfide gas. After 2 h, the reaction flask was heated to 65-70 °C for 1 h. The flask was allowed to cool, and then 100 ml of absolute ethanol were added before leaving the flask under nitrogen overnight. After this time, the solution was gravity-filtered to remove NaCl and then rotary-evaporated to remove ethanol. Recrystallization was performed with warm ethanol to yield a white solid with a melting point of 271.5 °C. The yield was 3.8 g (2.88 mmol).

Step 8: Iodoacetic Anhydride-- Iodoacetic acid (20 g, 107 mmol) was dissolved in 290 ml of ethyl acetate. Diisopropyl carbodiimide (6.8 g, 54 mmol) was added to this solution, and the reaction was stirred at room temperature for 1 h under nitrogen. IR spectroscopy showed that the reaction had gone to completion by the formation of two strong, sharp peaks at 1793.7 and 1735.6 cm¹ and the disappearance of a strong, broad peak at 3401.5 cm¹ (COOH peak). Ethyl acetate was removed by rotary evaporation, yielding a ruby red oil of iodoacetic anhydride.

Covalent Cysteine Modification with Biotin Reagents and Preincubations with -Bgtx or Erabutoxin a

Two days after transfection, cells were harvested by gentle agitation in phosphate-buffered saline containing 5 mM Na₂-EDTA (~0.5-1 × 10⁷ cells obtained from one 75-cm² tissue culture flask). After a brief centrifugation at ~600 × g, the cells were resuspended in high potassium Ringer's solution (22), pooled, divided into 300-µl aliquots, and incubated for the specified times with 5-500 µM MTSEDE-biotin or PEO-biotin. For MTSEA-biotin, the reagent was dissolved in Me₂SO instead of water before being added to the cells at 500 µM. For each biotin reagent, we added an excess of BrACh (1.5 mM) to terminate the reaction. The unbound biotin was removed by pelleting the cells (2 min at 20 °C), and resuspending the pellet in 0.5 ml of high potassium Ringer's. This wash was repeated three times in total. For preincubations with -Bgtx or Ea, the cells were incubated for 2 h at room temperature with 10 µM amounts of the respective toxin to allow for a saturation of binding sites. PEO-biotin was then added directly into the tubes for the times indicated. Typically, HEK-293 cells transiently transfected with wild-type AChR subunits yielded 50-100 fmol of biotinylated surface receptor/cm² of confluent cells.

Precipitation with Streptavidin-Agarose Beads

After treatment with PEO-biotin, cells were lysed in 450 µl of ice-cold RIPA solution (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with added proteinase inhibitors (10 µg/ml aprotinin, 200 µM phenylmethylsulfonyl fluoride, and 100 µM benzamide) and incubated on ice for 15 min. The lysate was centrifuged at 14,000 × g for 15 min, and the supernatant was precipitated with 50 µg of streptavidin-agarose beads (Pierce) at 4 °C overnight. Under these conditions, we found that the maximal amount of biotinylated receptor was precipitated, as the addition of larger volumes of beads did not lead to an increase in detectable -subunit. The precipitated samples were washed three times with 500 µl of RIPA solution (4,000 × g, 2 min, 4 °C). Typically, lysed cells containing ~1 mg of total protein were precipitated and the equivalent of ~200 fmol of toxin binding sites was retrieved and loaded onto SDS-polyacrylamide gels.

Surface Labeling with mAb 35 and Immunoprecipitation

To determine receptor surface expression, intact cells containing ~1 mg of total protein were incubated with 5 µg of monoclonal anti-nAChR mAb 35 (11, 23) in a final volume of 500 µl of Ringer's solution for 90 min on ice. Subsequently, unbound antibody was removed by washing as above, cells were lysed with 450 µl of RIPA solution as above, and receptors were precipitated overnight using 50 µl of Protein G-agarose. The precipitated samples were washed three times with 500 µl of RIPA solution (4,000 × g, 2 min, 4 °C). The conditions chosen are saturating, as neither the addition of larger amounts of Protein G-agarose or mAb 35 nor the prolonged exposure with mAb 35 lead to an increased precipitation of -subunit.

SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis

Biotinylated receptor-streptavidin-agarose bead complexes (or Protein G-receptor complexes in the case of mAb 35-treated samples) were brought to a final concentration of 4% SDS, 0.002% bromphenol blue, 0.12 M Tris-HCl, pH 6.8, and 10% glycerol. Prior to loading, DTT and -mercaptoethanol were added to a final concentration of 500 mM each. Samples were heated to 95 °C for 3 min before being loaded onto a 10% SDS-polyacrylamide gel (24). The gels were transferred onto polyvinylidene difluoride membranes and blocked with phosphate-buffered saline containing 0.1% Tween and 3% bovine serum albumin (Sigma). Primary antibody incubations were performed in the same buffer using a 1:500 dilution of antibody 43.37 (120 µg/ml stock) (25). Blots were then incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (working dilution 1:2500; Transduction Laboratories). Proteins were visualized using the enhanced chemiluminescence detection method (ECL Plus, Amersham Pharmacia Biotech).

Calculation of the Rates of Receptor Biotinylations

Visualized receptor bands were quantitated by densitometry using the software ImageJ (National Institutes of Health, Bethesda, MD). A calibration curve relating band intensities to femtomoles of ACh binding sites was established for mutant and wild-type receptors by running known concentrations of Torpedo membranes on a gel, followed by Western blotting, ECL exposure, and densitometry quantitation. These defined amounts of Torpedo membranes were run alongside with the biotinylated receptors and subjected to identical experimental conditions. Using these calibration curves, intensities of biotinylated receptor bands were then converted from pixel values to femtomoles of ACh binding sites for each time point. Rate constants for the biotinylation of AChR were then determined using a second-order rate equation (26). In cases where -Bgtx or Ea led to a substantial inhibition (>10-fold), second-order reaction rates were estimated by comparing the maximal amounts of biotinylation in the absence or presence of the toxins for three PEO-biotin concentrations (5, 50, and 500 µM).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In previous studies, we demonstrated that a cysteine can be substituted for all of the individual amino acids between positions 184 and 197 of the mouse muscle-type -subunit without dramatic effect on receptor functionality (<6-fold changes in the EC₅₀) as measured by ACh responsiveness in Xenopus oocytes (17). Moreover, we showed that within this region, residues Trp¹⁸⁷, Val¹⁸⁸, Phe¹⁸⁹, Tyr¹⁹⁰, and Pro¹⁹⁴ are solvent-accessible and are in a position to contribute to the -Bgtx binding site. In order to explore further the topology of the bracketing region extending from 184 to 197, we have now modified the appropriate Cys-substituted mutants with thiol-specific biotin derivatives following their expression in HEK-293 cells, permitting a more detailed analysis of the accessibility of these Cys-substituted residues.

PEO- and MTSEDE-biotin Specifically Modify Cysteines 192/193 in Reduced Wild-type nAChR-- Initially, we wanted to determine whether alkyl methane thiosulfonate derivatives of biotin could be used to specifically modify surface-exposed cysteines in the nAChR. Previously, we and others showed that smaller alkyl methane thiosulfonate derivatives and bromoacetylcholine, an alkylammonium compound containing an -haloacyl ester moiety, react covalently and specifically with Cys¹⁹² and Cys¹⁹³ of the nAChR following their selective reduction with 1 mM DTT (16, 17, 27-29). As shown in Fig. 2, when cells expressing the wild-type nAChR were selectively reduced at positions 192 and 193 with 1 mM DTT and then treated with 500 µM of MTSEDE-biotin (Fig. 2A, lane 4), we detected a band corresponding to the -subunit of the nAChR. As these studies were under way, a similar biotin derivative (PEO-biotin) became commercially available. When we applied this reagent to reduced wild-type receptors, we obtained results comparable to those for MTSEDE-biotin (Fig. 2B, lane 4). Importantly, this band was not present when unreduced nAChR was exposed to MTSEDE-biotin (Fig. 2A, lane 3) or PEO-biotin (Fig. 2B, lane 3).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Reactivity of wild-type nAChR toward various thiol-specific biotins. HEK-293 cells were transfected with  wild-type cDNA and the three other (, , ) AChR subunit cDNAs, harvested after 2 days of incubation, and treated with various thiol-specific biotin derivatives for 10 min at 20 °C. Streptavidin or Protein G precipitation (in the case of mAb 35), electrophoresis, Western blotting, and visualization of the -subunit were performed as described under "Materials and Methods." The concentration for all biotin reagents was 500 µM. Incubations for the individual lanes are as follows. A, lane 1, no DTT, no biotin; lane 2, 1 mM DTT (20 min, 20 °C); lane 3, MTSEDE-biotin; lane 4, 1 mM DTT (20 min, 20 °C), followed by three washes, followed by MTSEDE-biotin; lane 5, mAb 35 (see "Materials and Methods" for details); lane 6, MTSEA-biotin; lane 7, 1 mM DTT, followed by three washes, followed by MTSEA-biotin. B, lane 1, Torpedo membranes (100 fmol of -Bgtx binding sites); lane 2, 1 mM DTT; lane 3, PEO-biotin; lane 4, 1 mM DTT, followed by three washes, followed by PEO-biotin. Samples shown in panels A and B were run on separate gels. For each gel analysis, however, equivalent amounts of cell surface receptors (~200 fmol of toxin binding sites) were reacted with the respective biotin modifiers.

Biotinylation was blocked by preincubation with either 1.5 mM BrACh or 1.5 mM MTSET for both MTSEDE- and PEO-biotin, confirming its specificity (Fig. 3B, lane 3; Fig. 5A). In contrast, exposure of cells to the more hydrophobic MTSEA-biotin leads to considerable nonspecific labeling, as we detected -subunit labeling that was equally pronounced both before and after reduction with DTT. These results suggest that this reagent is capable of penetrating the lipid bilayer to a large degree and may have access to the internal cysteines at position Cys²²² (in presumed transmembrane segment M1) and Cys⁴¹⁸ (M4) (compare Fig. 2A, lanes 6 and 7). In addition, we observed that MTSEA-mediated biotinylation of reduced wild-type and mutant receptors cannot be blocked by 1.5 mM BrACh (data not shown), further pointing to its reactivity with an internal cysteine. For PEO- and MTSEDE-biotin, we detected nonspecific labeling of native unreduced receptors only when concentrations were raised to 1.5 mM and above (data not shown). The weak signal we obtained in the presence of DTT alone may suggest nonspecific absorption of the receptor to the streptavidin beads that are used for precipitation, although the result presented here was somewhat exceptional, and we generally did not observe a signal in the presence of DTT alone (Fig. 2A, lane 2). Additionally, labeling of surface-expressed -subunit by incubation of intact cells with the mouse monoclonal nAChR antibody, mAb 35 (11) (directed against region 67-76), followed by immunoprecipitation enabled us to the detect an -band of an intensity comparable to that for the biotinylated wild-type receptor, indicating that at least a large fraction of the surface population of wild-type nAChRs can be biotinylated (Fig. 2A, lane 5). However, it should be noted that a direct comparison of the signal intensities is not 100% accurate. Although we have optimized conditions such that maximal amounts of both mAb 35-labeled and biotinylated receptor are precipitated, it remains possible that the recovery of receptor eluted from the beads is different for the two methods. Nevertheless, we could use mAb 35 to test surface expression of all the cysteine mutants from position 184C to 197C, and we detected no noticeable difference in cell-surface expression levels among these mutants (Fig. 3A).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Surface expression of mutants 184C-197C. cDNAs encoding for wild-type or the respective mutant -subunits were transfected together with wild-type -, -, and -subunits into HEK-293 cells, which were harvested after 2 days of incubation, and treated with mAb 35 (A) or MTSEDE-biotin (B). Protein G and streptavidin precipitations, electrophoresis, Western blotting, and visualization of the -subunit were performed as described under "Materials and Methods." A, surface expression of mutant -subunit-containing nAChR in comparison to expression of wild-type receptor. For all the samples, equal amounts of receptor (~200 fmol in toxin binding sites) were exposed to mAb 35 as described under "Materials and Methods." For the lane denoted "Beads only," cells transfected with wild-type receptor were used, lysed without prior mAb 35 treatment, and precipitated overnight with Protein G-agarose beads. B, biotinylation of mutant F189C with MTSEDE-biotin can be blocked by BrACh pretreatment. For lanes 2 and 3, the incubation with 500 µM MTSEDE-biotin was performed for 10 min at 20 °C, and terminated by the addition of 1.5 mM BrACh. For lane 3, 1.5 mM BrACh was first added for 10 min at 20 °C, followed by three washes. Then, MTSEDE-biotin was added. Lane 1, no reagent added; lane 2, MTSEDE-biotin only; lane 3, BrACh and MTSEDE-biotin; lane 4, mAb 35 only (as in A). For all the samples, equal amounts of receptor (~200 fmol in toxin binding sites) were modified and loaded into each lane.

Rate of Biotinylation of Reduced Wild-type nAChR and Retardation in the Presence of -Bgtx-- In an effort to quantitate the reactivity of reduced wild-type and mutant nAChRs with PEO-biotin, we exposed nAChRs to 5 µM PEO-biotin for various incubation times (Fig. 4A). Second-order reaction rates were calculated as described under "Materials and Methods." For reduced wild receptors, we obtained a rate constant of 103.6 M¹ s¹ (Table I). Comparable results were obtained with MTSEDE-biotin (data not shown). However, since the MTS compound was more light-sensitive and degraded rapidly at room temperature, we focused the remainder of our studies exclusively on the effects mediated by PEO-biotin.

View this table: [in this window] [in a new window]   Table I Second-order reaction rates of mutant -subunits with PEO-biotin Wild-type or the respective mutant -subunits encoding cDNAs were transfected together with wild-type -, -, and -subunits into HEK-293 cells, which were harvested after 2 days of incubation, and treated with PEO-biotin. Streptavidin precipitation, electrophoresis, Western blotting and visualization of the -subunit and calculation of second-order reaction rates were performed as described under "Materials and Methods." Second-order reaction rate constants in the absence (k) and presence of -Bgtx (kBgtx) were determined using 50 µM PEO-biotin for all of the mutants, except for mutant W187C (500 µM) and reduced wild-type receptors (5 µM). Positions 192 and 193 form a vicinal disulfide bond in the wild-type receptor and are not listed separately, since they are indistinguishable and become modified in PEO-biotin-exposed reduced wild-type receptors. In cases where -Bgtx led to a blockade of the second-order reaction rate with PEO-biotin, only estimates of the maximal rates of biotinylation in the presence of -Bgtx could be calculated, due to nonspecific labeling at high concentrations of PEO-biotin. The reduction in the rate of reactivity was estimated by comparing the intensities obtained for 10-min incubations with 5, 50, and 500 µM biotin in the presence and absence of the -Bgtx.

When 10 µM -Bgtx was added to cells and allowed to equilibrate for 2 h, the reactivity of reduced wild-type receptors toward PEO-biotin was substantially slowed (>100-fold). Using 5 and 50 µM PEO-biotin, we could not detect any biotinylation of the -subunit, and even after incubation with 500 µM PEO-biotin, we obtained only a weak signal (~10% of the maximum intensity; Fig. 4B). A precise determination of the second-order rate constant in the presence of -Bgtx was not possible, since PEO concentrations of 1.5 mM and above would have to be used, and under these conditions, there was considerable background stemming from the presumed modification of an internal cysteine or other unidentified sites.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Reactivity of reduced wild-type receptors with PEO-biotin and inhibition of biotinylation in the presence of -Bgtx. Wild-type -subunit encoding cDNAs were transfected together with wild-type -, -, and -subunits into HEK-293 cells, which were harvested after 2 days of incubation, and treated with PEO-biotin. The PEO-biotin concentration for all time points was 5 µM, unless otherwise indicated in the figure. Streptavidin precipitation, electrophoresis, Western blotting, and visualization of the -subunit were performed as described under "Materials and Methods." A, time course of biotinylation in the absence of -Bgtx. Biotinylation was terminated by the addition of the thiol-specific BrACh (1.5 mM) at the indicated time points. B, biotinylation in the presence of 10 µM -Bgtx. Harvested cells were first incubated with 10 µM -Bgtx for 2 h, and PEO-biotin was then added directly. In lane 1, Torpedo membranes (100 fmol of toxin binding sites) were loaded as a standard reference. For all samples in A and B, transfected cells were pooled prior to modification, and equal amounts of receptor (~200 fmol of toxin binding sites) were modified and loaded onto each lane.

Rate of the Biotinylation of Mutant V188C nAChR Is Unaffected by the Presence of -Bgtx-- Previous results showed that mutant V188K leads to a ~680-fold decrease in affinity for a short -neurotoxin, NmmI, although the effect of this mutation was much less pronounced in affecting -Bgtx binding (15, 30, 31). In addition, modification of V188C with 1.5 mM BrACh or MTSET leads to a ~50% reduction in the number of -Bgtx binding sites (16). On the other hand, a mutation introducing a negative charge at this position (V188D) merely produced a ~10-fold decrease in NmmI binding. To more closely examine the role of this position in -Bgtx binding, we determined second-order reaction rates for V188C with 50 µM PEO-biotin in the presence (Fig. 5B) and absence of -Bgtx (Fig. 5A). In the absence of -Bgtx, we obtained a rate of 102 M¹ s¹, whereas incubation in the presence of -Bgtx resulted in only a slightly diminished rate of 77 M¹ s¹. Therefore, we found no indication that -Bgtx binding to the -subunit protected this Cys-substituted residue from biotinylation. Interestingly, a comparison between Figs. 4 and 5 shows that the signal intensity for the biotinylation of mutant V188C is less than that for reduced wild-type receptors, despite the fact that their reaction rates are comparable (Table I). Most likely, two factors contribute to this observation. First, reduced wild-type receptors contain two modifiable free thiol groups (Cys^192/193), i.e. although the reaction rate is comparable to that for V188C, the signal intensity will be twice as high for any given time point. Additionally, it is possible that, for V188C, only a portion of the receptor population contributes to the signal, whereas the rest is either refractory to biotinylation or to the subsequent sterically constrained reaction with streptavidin beads. This view is strengthened by the fact that preincubation with -Bgtx enhances the yield of biotinylated receptor approximately 2-fold (Fig. 6C).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Reactivity of mutant V188C with PEO-biotin in the presence or absence of -Bgtx. The V188C mutant -subunit encoding cDNA was transfected together with wild-type -, -, and -subunits into HEK-293 cells, which were harvested after 2 days of incubation, and treated with PEO-biotin. The PEO-biotin concentration for all time points was 50 µM, unless otherwise stated in the figure. Streptavidin precipitation, electrophoresis, Western blotting, and visualization of the -subunit were performed as described under "Materials and Methods." A, biotinylation in the absence of -Bgtx. Biotinylation was terminated by the addition of the thiol-specific BrACh (1.5 mM) at the indicated time points. Torpedo membranes (100 fmol in toxin binding sites) were loaded as a standard reference. For BrACh preincubation, the sample was exposed to 1.5 mM BrACh for 10 min at 20 °C, followed by three washes, prior to the addition of PEO-biotin. B, biotinylation in the presence of 10 µM -Bgtx. Harvested cells were first incubated with 10 µM -Bgtx for 2 h, and PEO-biotin was then added directly. For all samples in A and B, equal amounts of receptor (~200 fmol of toxin binding sites) were modified and loaded onto each lane.

View larger version (16K): [in this window] [in a new window]   Fig. 6.   Protection of PEO-biotin-mediated modification of reduced wild-type nAChR and mutants W187C, V188C, and F189C in the presence of -Bgtx or Ea. Wild-type or the respective mutant -subunits encoding cDNAs were transfected together with wild-type -, -, and -subunits into HEK-293 cells, which were harvested after 2 days of incubation, and treated with PEO-biotin at the concentrations indicated. Streptavidin precipitation, electrophoresis, Western blotting, and visualization of the -subunit were performed as described under "Materials and Methods." A, protection of the biotinylation of reduced wild-type receptors by -Bgtx and Ea. Wild-type receptor was reduced with 1 mM DTT (20 min, 20 °C). All biotinylations were done with 5 µM PEO-biotin for 10 min at 20 °C. For lanes 3 and 4, 10 µM -Bgtx or Ea, respectively, were added for 2 h prior to PEO-biotin addition. Lane 1, Torpedo membranes (200 fmol in toxin binding sites); lane 2, PEO-biotin only; lane 3, -Bgtx and PEO-biotin; lane 4, Ea and PEO-biotin. For lanes 2-4, equal amounts of receptor (~200 fmol in toxin binding sites) were reacted and loaded. B, protection of the biotinylation of mutant W187C by -Bgtx and Ea. For all the lanes shown, biotinylations were performed with 500 µM PEO-biotin for 10 min at 20 °C. Lane 1, Torpedo membranes (200 fmol in toxin binding sites); lane 2, PEO-biotin only; lane 3, -Bgtx (10 µM) and PEO-biotin; lane 4, Ea (10 µM) and PEO-biotin. For lanes 2-4, equal amounts of receptor (~100 fmol in toxin binding sites) were reacted and loaded. C, protection of the biotinylation of mutant V188C by -Bgtx and Ea. For all the lanes shown, biotinylations were performed with 50 µM PEO-biotin for 10 min at 20 °C. Lane 1, Torpedo membranes (200 fmol in toxin binding sites); lane 2, PEO-biotin only; lane 3, -Bgtx (10 µM) and PEO-biotin; lane 4, Ea (10 µM) and PEO-biotin. For lanes 2-4, equal amounts of receptor (~100 fmol in toxin binding sites) were reacted and loaded. D, protection of the biotinylation of mutant F189C by -Bgtx and Ea. All biotinylations were performed with 50 µM PEO-biotin for 10 min at 20 °C. Lane 1, PEO-biotin only; lane 2, -Bgtx (10 µM) and PEO-biotin; lane 3, Ea (10 µM) and PEO-biotin; lane 4, Torpedo membranes (200 fmol in toxin binding sites). For lanes 1-3, equal amounts of receptor (~100 fmol in toxin binding sites) were reacted and loaded.

Evidence That All Amino Acid Residues within Region 184 to 197 Are Solvent-exposed-- Although positions W187C, V188C, F189C, Y190C, and P194C were modifiable with BrACh and their modification resulted in a substantial blockade in -Bgtx binding (16), the surface disposition of the other residues spanning region 184 to 197 is not well understood. Specifically, it was previously impossible to distinguish whether cysteine-substituted residues 184-186, 191, and 195-197 were modified following the application of thiol-specific reagents. By systematically examining the reactivity of these residues with PEO-biotin, we now show that all of these residues are surface-exposed and accessible to biotinylation to a similar degree. Their second-order reaction rate constants fell within the range of ~10 to ~100 M¹ s¹ (W187C: 10.8 M¹ s¹; V188C: 101.9 M¹ s¹; Table I).

Table I also lists the second-order reaction rate for positions D71C and W184C to P197C in the presence of saturating concentrations of -Bgtx. Importantly, the reaction rate for the biotinylation of D71C is unaffected in the presence of -Bgtx, confirming its location outside of the -Bgtx binding site (11). Apart from reduced wild-type receptors, only residues W187C (at least a 10-fold reduction) and F189C (at least a 100-fold reduction) exhibit a substantial reduction in the reaction rate in the presence of -Bgtx. It should be noted that an accurate determination of reaction rates for these mutants in the presence of -Bgtx was not possible. This would have required using concentrations of PEO-biotin in excess of 1.5 mM, a concentration that leads to the modification of other sites in addition to the engineered cysteine. Due to its reduced reactivity with PEO-biotin even in the absence of -Bgtx, this is especially true for mutant W187C.

Effects of Erabutoxin a Co-incubation on the Reactivity of Residues W184C to P197C with PEO-biotin-- Previous studies suggested contradicting roles for positions Trp¹⁸⁷, Val¹⁸⁸, and Phe¹⁸⁹ in the binding of -neurotoxins. For example, using a double mutant cycle analysis of the short-chain -neurotoxin NmmI with nAChRs, Ackermann et al. (31) found no indication for the involvement of Trp¹⁸⁷ and Phe¹⁸⁹ in toxin binding. On the other hand, our previous work suggests that all three positions (187-189) contribute to the binding of the long-chain -neurotoxin -Bgtx. To characterize further the precise roles of these residues, we expanded our biotinylation studies to investigate the effect of a short -neurotoxin. In this assay, Erabutoxin a (10 µM) was added to HEK-293 cells transfected with W187C, V188C, and F189C 2 h prior to PEO-biotin addition.

Fig. 6 shows the profile of -neurotoxin (-Bgtx or Ea) protection from biotinylation for reduced wild-type, W187C, V188C, and F189C mutant receptors. Biotinylation of reduced wild-type receptors is blocked by approximately 90% with Ea and completely with -Bgtx (Fig. 6A, lanes 2-4) when 5 µM PEO-biotin is added for 10 min. In contrast, Ea does not protect positions W187C and F189C from biotinylation (Fig. 6, B (lanes 2 and 4) and D (lanes 1 and 3), respectively). -Bgtx, on the other hand, clearly blocks biotinylation at these positions (Fig. 6, B (lane 3) and D (lane 2)). Similarly, Ea completely abrogates biotinylation of mutant V188C (Fig. 6C, lanes 2 and 4), whereas -Bgtx has little effect (Fig. 6C, lane 3). It should be noted that different PEO-biotin concentrations were used to account for the different reaction rates of the various mutants. However, we repeated these experiments for reduced wild-type nAChRs with 50 and 500 µM PEO-biotin and for V188C and F189C with 500 µM and obtained comparable results (data not shown).

For all other positions, the effects of Ea and -Bgtx on PEO biotinylation of engineered cysteines seem to be comparable; none of the other positions shows a substantial blockade of biotinylation, as is summarized in Table II. Again, as stated above (and in the legend for Tables I and II), accurate determinations of second-order rate constants for the biotinylation in the presence of Ea were not possible for positions W187C, V188C, and reduced wild-type receptors. Furthermore, in those cases where Ea did not appear to protect against biotinylation, precise reaction rates were not calculated. Instead, the reduction of the rate was estimated by comparing the intensities obtained for 10-min incubations with 5, 50, and 500 µM PEO-biotin in the presence and absence of Ea.

View this table: [in this window] [in a new window]   Table II Comparison of the inhibition of substituted cysteine biotinylation by -Bgtx and erabutoxin a Wild-type or the respective mutant -subunits encoding cDNAs were transfected together with wild-type -, -, and -subunits into HEK-293 cells, which were harvested after 2 days of incubation, and treated with PEO-biotin. Only region 187 to 193 is shown, since there was no difference in the observed effects for -Bgtx and Ea for the other mutants examined. Results for positions 192 and 193 were obtained with reduced wild-type receptor, which contains two free thiols at these positions. The symbol "+" in the table denotes a greater than 10-fold reduction in the second-order reaction rate constant k in the presence of either -Bgtx or Ea. The symbol "" is used to represent a change of less than 2-fold in the reaction rate (for -Bgtx), or a <50% reduction in the maximum amount of labeling in the presence of Ea. For reasons described in the text and Table I, rates for the biotinylation of mutants W187C, F189C, and Cys192/193 in the presence of -Bgtx are estimates of the maximum rate and could not be determined accurately from the data. This is also the case for the biotinylation of positions V188C and Cys192/193 in the presence of Ea.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Our primary aim was to define in more detail the topology and solvent accessibility of residues 184-197 in the -subunit of the mouse muscle nicotinic acetylcholine receptor. To achieve this, we engineered individual cysteine substitutions and modified the introduced cysteine side chains with water-soluble, thiol-reactive derivatives of biotin. A similar approach has been used, for example, by Grunewald and co-workers (32) to determine the topology of the astroglial glutamate transporter GLT-1. Likewise, the solvent-accessible structure of the -aminobutyric acid A receptor has been investigated with a combined cysteine mutagenesis/biotinylation procedure (33).

Thiol-reactive Biotin Derivatives That Are Hydrophilic Are More Selective in Modifying External Cysteines-- The success and accuracy of cysteine modifications with thiol-reactive biotin derivatives strongly depends on the hydrophilicity of the reagent. As can be seen in Fig. 1 (A and B), the relatively hydrophobic modifier MTSEA-biotin reacts strongly with wild-type nAChR. In these receptors, there are intrinsic cysteine disulfide pairs at positions 128 and 142 as well as at positions 192 and 193, but no free thiols in the extracellular domain, and thus there should be no reactivity with thiol reagents. The observed labeling is most likely explained by MTSEA-biotin entering the lipid bilayer and reacting with the solvent inaccessible unpaired Cys²²² and/or Cys⁴¹⁸. This is further supported by the fact that preincubation with 1.5 mM BrACh does not protect against biotinylation of reduced wild-type and mutant receptors when MTSEA-biotin is used (data not shown). In sharp contrast, the more hydrophilic, water-soluble derivatives, MTSEDE-biotin and PEO-biotin, do not react with wild-type nAChR unless the disulfide bond at positions 192/193 is selectively reduced with 1 mM DTT (27, 28), indicating that these reagents are not membrane-permeable. With the hydrophilic reagents, biotinylation was blocked completely using either 1.5 mM BrACh or MTSET (see Fig. 3B for MTSEDE-biotin and Fig. 5A for PEO-biotin). As BrACh and MTSET are charged derivatives, their block of biotinylation confirms the specificity of the reaction and supports the conclusion that biotinylation with MTSEDE-biotin or PEO-biotin is restricted to the solvent-accessible surface of the nAChR in our studies using intact cells. Either biotin conjugate would have been useful in the present study, although we decided to concentrate on PEO-biotin, as MTSEDE-biotin was more light-sensitive and degraded more rapidly at room temperature. Nonetheless, MTSEDE-biotin would offer advantages in cases where the introduction of a reversible disulfide bond is desired.

All Amino Acid Residues between Positions 184 and 197 Are Solvent-exposed-- Table I summarizes the second-order reaction rates for the individual Cys substitutions of amino acid residues between positions 184 and 197. Their second-order reaction rate constants fall within the range of ~10 to ~100 M¹ s¹ (W187C: 10.8 M¹ s¹; V188C: 101.9 M¹ s¹). As even the least reactive of these positions, W187C, clearly contributes to the -Bgtx binding site and is modifiable by the charged reagent BrACh (16), we conclude that second-order reaction rates for modification with PEO-biotin that are as low as 10 M¹ s¹ are consistent with solvent accessibility. We further conclude, therefore, that all of the residues within the region tested are surface-exposed. Minor perturbations in reactivity occur, in general, over a 10-fold range and are likely to be due to variations in the chemical and steric environment surrounding the individual positions (34). Additionally, favorable electrostatic and steric interactions may enhance biotinylation. As an additional reference point, we included an analysis of mutant D71C. It is well established that Asp⁷¹ forms one of the main determinants in the epitope recognized by antibodies directed against the main immunogenic region (11) and therefore should be surface-accessible. Indeed, the second-order reaction rate (39.5 M-1 s¹) is also in the range obtained with the 184-197 series of Cys substitutions. Similarly, a comparable reaction rate constant (~200 M¹ s¹) has been reported for the reaction of an N-ethylmaleimide derivative with -mercaptoethanol in phosphate buffer (35).

Further, the second-order reaction rate with PEO-biotin seems to be identical for the two -subunits of the nAChR, regardless of position of the introduced cysteine mutation. Using a linear regression fitting routine, we calculated a curve with a very good fit to the data (R > 0.98 for all the mutants investigated; data not shown) assuming a single class of binding sites.

Our conclusions on the solvent accessibility of the 184-197 region are in line with a number of recently published studies. Structural predictions derived from the NMR studies of a receptor-peptide fragment bound to -Bgtx (36) suggest that residues 187-190 are likely to be surface-accessible in the native receptor. The conclusions concerning surface accessibility of -subunit residues 187-190 are also consistent with studies of the Bgtx-resistant nAChRs found in cobra and mongoose muscle and of HEK-expressed mouse muscle nAChRs containing glycosylation signals found in the cobra and mongoose AChR (23, 37). The surface accessibility of Val¹⁸⁸ was demonstrated by McLaughlin et al. (17) and is also supported by recent studies of Ackermann et al. (30, 31). A recent double mutant cycle analysis concludes that position Pro¹⁹⁷ interacts with NmmI residues Arg³³ and Lys²⁷ and must therefore be surface-exposed. In addition, our study demonstrates for the first time that positions corresponding to Trp¹⁸⁴, Lys¹⁸⁵, Ser¹⁹¹, Thr¹⁹⁵, and Thr¹⁹⁶ are on the solvent-accessible surface of the receptor.

-Bgtx Protects Mutants W187C, F189C, and Reduced Wild-type Receptors, but Not Mutant V188C, from Biotinylation with PEO-biotin-- In the present study, we provide evidence that positions 71, 184, 185, 191, 195, 196, and 197 do not form part of a stable -Bgtx binding site, as the presence of bound -Bgtx does not have a significant effect on the second-order reaction rate for PEO-biotin (Table I). One reservation concerning the interpretation of the Cys substitution studies is that the substitution itself may locally distort the structure. Thus, negative results of the protection experiments may not be as conclusive as positive results. Furthermore, it is possible, depending on the local geometry, that an introduced cysteine is both in some proximity to bound Bgtx and in a position where Bgtx does not occlude the site from biotinylation.

Interestingly, the biotinylation of mutants V188C and Y190C is not affected by the presence of -Bgtx, even though the tethering of a methylammonium moiety to these residues leads to a blockade of -Bgtx binding (16). These results allow us to refine our understanding of the interaction between -Bgtx and positions 188 and 190, as they suggest that these positions, at least when substituted with cysteine side chains, do not interact directly and stably with -Bgtx (Fig. 3 and Table I). Nevertheless, there is good evidence that these positions are within 8 Å of the toxin binding site. The introduction of an alkylammonium moiety, through the action of either BrACh or MTSET, leads to a significant perturbation of the receptor-toxin interaction (16). With both BrACh and MTSET, a covalently attached adduct of cysteine is formed that would fit into a cylinder 8 Å long and 6 Å in diameter (38). Furthermore, the results presented here are compatible with NMR structural studies of the complex formed between the dodecapeptide 185-196 and -Bgtx (36). The NMR analysis revealed intermolecular nuclear Overhauser effect cross-peaks between one -methyl group of Val¹⁸⁸ and the two -methyl groups of Bgtx residue Val³⁹, thus placing these latter methyl protons at a distance of ~4-5 Å from that of Val¹⁸⁸. Although this distance constraint certainly places Val¹⁸⁸ in the proximity of Bgtx residue Val³⁹ in this complex, there was no further indication of a more intimate or extensive contact between these hydrophobic side chains. In contrast, -Bgtx clearly blocks the biotinylation of residues W187C and F189C, and thereby provides independent support that these residues are critical for a stable receptor-toxin interaction (16, 37, 39). Furthermore, the biotinylation of positions 192 and 193 in reduced wild-type receptors is also inhibited by -Bgtx and underlines the importance of these residues for -Bgtx binding. Nevertheless, the modification of these cysteines and of W187C and F189C with non-methylammonium-containing modifiers does not affect -Bgtx binding significantly (16), suggesting additional complexity in Bgtx binding. In addition, it should be emphasized that the saturation binding assay used in these modification studies would not have detected decreases of -Bgtx binding affinity <50-fold (16). Theoretically, it is also possible that the presence of -Bgtx leads to the complete abrogation of biotinylation on only one of the two -subunits, whereas the other site remains unaffected. If this were the case, we would expect to see a 2-fold reduction of the maximum intensity of the biotinylation signal. Our results argue against such a scenario, since the maximum intensity of the signal is largely unaltered for all of the investigated mutants.

It is somewhat surprising that the "footprint" of -Bgtx protection from biotinylation is not larger. Previous studies have suggested that the Bgtx-receptor contact site would involve multiple points and a large portion of the toxin surface (40, 41). Our results suggest that the number of strong contacts may be fewer than expected. In addition, any additional contacts may be flexible enough to allow sufficient structural fluctuation to permit access to the reactive derivatives over the time course of the reaction incubation. Alternatively, some of the residues in this region may interact with Bgtx and the biotinylation reagents via non-overlapping surfaces.

Short and Long Chain Neurotoxins Establish Distinct Contact Points with the nAChR-- Ackermann et al. (31) have argued that the short neurotoxin NmmI interacts with Val¹⁸⁸, whereas their results do not implicate positions Trp¹⁸⁷ and Phe¹⁸⁹ in NmmI binding. The results presented here (Fig. 6, Table II) seem to reconcile the NmmI studies with our previous results, suggesting an important role of Phe¹⁸⁹ in Bgtx binding (16, 39). Second-order reaction rates for the biotinylation of positions W187C and F189C are inhibited at least 10-fold by bound -Bgtx, whereas V188C is not affected. In contrast, Ea, a short neurotoxin very similar to NmmI, leads to a >10-fold protection of position V188C, but does not alter biotinylation at positions W187C and F189C. This suggests that -Bgtx and Ea interact with distinct amino acids on the nAChR.

A Model for the Bgtx-mediated Blockade of Receptor Biotinylation-- The model we propose here for the mechanism of -Bgtx-mediated blockade of biotinylation is based on the mode of interaction between fasciculin and acetylcholine esterase (42-47). In this model, -Bgtx, which carries a large net positive charge (+4; see Refs. 48 and 49) could bind to a site near a gorge leading to the agonist binding site and could obstruct PEO-biotin access to substituted cysteines (or ACh access to its binding site), which, in our experiments, would be reflected by a decrease in the second-order reaction rate of biotinylation. It is unlikely, however, that -Bgtx blocks t