Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The binding sites for agonists and competitive antagonists in the nicotinic acetylcholine receptor (nAChR) are within the extracellular domain at the - and - subunit interfaces. Affinity labeling and mutagenesis studies have provided extensive evidence for a model of the agonist site structure with contributing amino acids from three distinct regions of the -subunits (referred to as binding site segments A, B, and C) and from at least three regions of the  (or )-subunit (segments D, E, and F) (reviewed in Prince and Sine, 1998; Arias, 2000; Corringer et al., 2000). Most features of the model are present in the binding site identified within the recently solved structure of a molluscan, glial-derived soluble ACh binding protein (AChBP), a homopentameric structural and functional homolog of the N-terminal ligand binding domain of a nAChR -subunit (Brejc et al., 2001; Smit et al., 2001).

The substituted Cys accessibility method (Karlin and Akabas, 1998) has provided an alternative approach for characterizing structural features of the nAChR and other ion channels. An observed irreversible change in the functional properties of the channel, after exposure to a water-soluble sulfhydryl reagent, suggests that the substituted Cys is exposed at the water accessible protein surface. This technique has been used to identify the state-dependent accessibility of amino acids contributing to the ion conduction pathway of the nAChR (Akabas et al., 1994; Akabas and Karlin, 1995; Zhang and Karlin, 1997, 1998). In studies of the structure of the agonist binding site, which contains a disulfide bond between Cys-192/193 in segment C, most Cys substitutions are well tolerated within 184-198 and are accessible for modification (McLaughlin et al., 1995; Spura et al., 1999; Spura et al., 2000). Using Cys mutagenesis of Torpedo californica nAChR, we previously tested the accessibility of positions identified by affinity labeling and mutagenesis in segments A (Tyr-93), B (Trp-149), C (Tyr-190 and Tyr-198), and D (Trp-55 and Glu-57) as well as surrounding amino acids in segments A (90-96) and D (52-58) and found that Tyr-93, Asn-94, Tyr-198, and Glu-57 were accessible (Sullivan and Cohen, 2000). That study also helped to define the structural requirements for ligand orientation compatible with nAChR activation, as [2-(trimethylammonium)-ethyl]-methanethiosulfonate (MTSET), which attaches thiocholine, acted as an irreversible antagonist at positions Y93C and E57C but as a covalent agonist at Y198C. Furthermore, a structural analog with the tethering arm shortened by one methylene group (0.7 Å) acted as an irreversible antagonist at Y198C and at all other accessible positions.

In this report, we extended these studies by identifying additional accessible residues in segment C (195-201) as well as segment E (106-113), which includes residues identified by photoaffinity labeling with the antagonists [³H]4-benzoylbenzoylcholine (Leu-109; Wang et al., 2000) and [³H]d-tubocurarine (dTC) (Tyr-111; Chiara et al., 1999). With these mutant nAChRs, we also tested whether MTSET or its analogs could act as irreversible agonists when tethered at positions other than Y198C. In addition, we used the panel of nAChR binding site mutants containing accessible cysteines to identify positions that could be protected from alkylation when the agonist binding site was occupied by dTC. Based upon photoaffinity labeling, [³H]dTC binds to the agonist site at the - interface near amino acids in segments C (Tyr-190, Cys-192, and Tyr-198), D (Trp-55), and E (Tyr-111 and Tyr-117; Chiara and Cohen, 1997; Chiara et al., 1999), and dTC can protect against MTSET reaction at Y198C (Sullivan and Cohen, 2000). The results we now report, which indicate protection of alkylation of positions in segments C, D, and E but not in segment A, are consistent with the results of photoaffinity labeling. The pattern of protected residues within segment C indicates that this region is organized as a -strand and identifies a surface projecting toward the ACh binding site consistent with the structure of the binding site in the AChBP (Brejc et al., 2001). However, the lack of protection of Y93C is surprising in terms of that structure. Our experimental results are compared with a homology model of the T. californica agonist binding site based on the crystal structure of the AChBP and are generally consistent with this structure. However, the accessibility of some residues suggests differences between the model and the structure of the T. californica nAChR binding site in the absence of agonist (our experimental conditions).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

cDNA Mutagenesis. Mutants were constructed by "overlap extension" PCR using T. californica nAChR subunit plasmids (, , and  in pMXT and  in pSP64) and reagents as described previously (Sullivan and Cohen, 2000). The mutations of amino acids 195-201 of the mature -subunit were generated using primers which gave a PCR product of ~1000 base pairs that could be subcloned using the unique BsiWI restriction site and the BbvII site near the 3' end of the -subunit coding region. Mutations of amino acids 106 to 113 in the mature -subunit were made by generating a PCR product of ~1.13 kilobases that was subcloned using the unique HindIII site in the vector and the StuI site in the -subunit coding region. Each PCR mix contained 0.5 µM primers, 50 ng of template DNA, and 0.4 mM dNTPs in the reaction buffer supplied with the enzyme. PCR reactions were for 24 cycles with a three-step protocol (1.5 min at 95°C, 45°C, and 72°C).

Electrophysiology. T. californica nAChR subunit-specific cRNAs were transcribed in vitro and Xenopus laevis oocytes were injected as described previously (Sullivan and Cohen, 2000). Isolated, follicle-free oocytes were injected with 0.5 to 10 ng of subunit-specific RNAs in a molar ratio of 2///, and currents elicited by ACh were measured 48 to 72 h after injection by two-electrode voltage clamp. Under our experimental conditions, for oocytes injected with 0.5 ng of wild-type nAChR subunit cRNAs, maximal current responses for ACh were typically 1 to 2 µA. For the Cys substitutions between 195 and 201 in segment C, the maximal current responses for ACh were similar to wild-type for the mutant nAChRs containing Cys at 195, 196, 197, or 199. As described previously, the Y198C nAChRs showed maximal current levels ~1% of wild-type, and for the D200C and I201C mutant nAChRs, the maximal currents were also 1 to 5% of wild-type. For Y198C, as judged by binding of ¹²⁵I--bungarotoxin to intact oocytes, surface nAChR levels were ~50% of wild-type. Within segment E (106-113), the maximal ACh current responses were similar to wild-type for each substitution except for Y111C, which had maximal responses ~2% of wild-type and surface nAChR levels <10% of wild-type. Surface receptor expression levels of other mutants were not quantified. Salts, atropine, ACh, and dTC were from Sigma (St. Louis, MO). MTSET, [3-(trimethylammonium)-propyl]-methanethiosulfonate (MTSPT), 2-aminoethylmethanethiosulfonate (MTSEA), and 4-(N-maleimido)benzyltrimethylammonium (MBTA) were from Toronto Research Chemicals (North York, Ontario, Canada), and Biotin-PEO-maleimide (Fig. 1) was from Pierce (Rockford, IL). Sulfhydryl-modifying reagents were prepared as millimolar stock solutions in recording solution and stored on ice during use, with fresh solutions prepared approximately every 2 h. For nAChR activation, ACh dose response curves were fit to the equation: I / I_max = [1 + (K_app / [ACh])^n_H]¹, where I and I_max are the currents at a given concentration of ACh and the maximal current, respectively. K_app is the apparent activation constant for ACh and n_H is the Hill coefficient. pCLAMP (Axon Instruments, Foster City, CA) and SigmaPlot (SPSS Inc., Chicago, IL) software were used for data analysis.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Structures of sulfhydryl-modifying reagents. The extended length of ACh measures 8.7 Å. Reaction with MTSEA transfers to the Cys sulfur the primary amine 2-aminoethanethiol, which, in an extended conformation, extends 5.8 Å from the point of attachment to the surface of the primary amine group. Reaction with MTSET transfers thiocholine, which can extend 6.9 Å from the Cys sulfur to the surface of the trimethylammonio group. MBTA positions the trimethylammonio group 12.2 Å from the point of attachment. Biotin-PEO-maleimide has an extended length of 29 Å.

Rate Constants of nAChR Modification. For the sulfhydryl-reactive reagents producing irreversible inhibition of ACh responses, the time course of the reaction with a substituted Cys mutant in the absence of ACh was determined by recording the initial response to ACh and then the response to ACh after repeated applications of modifying reagent for 5-s intervals. Each application of reagent was followed by a ~1-min wash, three ACh test applications (5 s each), and a 1-min wash. ACh was generally applied at a concentration equal to K_app. ACh-induced currents after treatment were plotted as a function of cumulative modification time (t) and fit by a single exponential function, I_t = I + (I_o  I) exp(t / ) where I_t is the current at a given time, I is the amount of current remaining after the reaction is complete, and I_o is the initial current level. 1 /  is the pseudo-first-order rate constant and the second-order rate constant, k, is (1 / ) / x, where x is the concentration of modifying reagent.

dTC Protection Assay. Responses to ACh at a concentration near K_app were measured before and after coapplication of dTC and ACh to show that 10 µM dTC was sufficient to reversibly block >95% of the ACh response and that the Cys substitution itself had not interfered with the receptors' ability to bind dTC. This initial part of the assay was also necessary to determine that the effects of dTC were reversible. It was often necessary to wash the oocyte for several minutes after dTC application for full recovery of the ACh response. To measure the degree of protection by dTC, ACh test pulses were measured before and after 10 µM dTC was coapplied with a concentration of MTSET known to cause 50 to 80% inhibition (based on rate constants). The same concentration of MTSET was then applied in the absence of dTC, again using ACh test pulses to measure the extent of inhibition. The degree of protection was then determined by comparing the ratio of the extent of modification in the absence of dTC to the extent of modification in the presence of dTC: % protection = [1  (% Inhibition_dTC/MTSET / % Inhibition_MTSET)] × 100.

Homology Modeling of the T. californica nAChR. Molecular modeling of the extracellular domain of the T. californica nAChR based upon the recently published structure of the AChBP (Brejc et al., 2001) was done using Insight II (Version 98; MSI, San Diego, CA) on a Silicon Graphics O₂ workstation. The sequences for the four T. californica nAChR subunits (NCB accession numbers: ACRYA1, ACRYB1, ACRYG1, and ACRYD1) were obtained from the National Center for Biotechnology Information and the coordinates for the structure of the AChBP (PDB number 1I9B) were obtained from the Research Collaboratory for Structural Bioinformatics (http://www.rcsb.org). The nAChR subunit sequence alignment presented by Brejc et al. (2001) was used, and the AChBP structure was examined to ensure that insertions and deletions occurred within exposed flexible segments. The Insight II Homology module placed the nAChR sequences into the AChBP structure and the Insight II Discovery module energy minimized the resulting structural model. Compared with the primary structure of the AChBP, the nAChR -subunit contains insertions in two regions within the ACh binding site. Although segment C in all neuronal nAChR -subunits aligns well with the AChBP, the T. californica and skeletal muscle -subunits contain a single residue insertion between Cys-193 and Tyr-198. We designated Asp-195 as the inserted residue to preserve the 10-strand beginning at Thr-196, with Thr-196, Tyr-198, and Asp-200 projecting in the direction of the binding site, a structure consistent with experimental results described in this report. The other -subunit insertion in the vicinity of the binding site was Ala-96 in segment A. Both insertions were well tolerated upon minimization.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Functional Properties of nAChRs with Cys Substitutions in Binding Site Segments C and E. nAChR functional properties were assessed by measuring ACh-elicited currents using two-electrode voltage clamp. Each of the substitutions within 195-201 and 106-113, when expressed with other wild-type subunits, resulted in functional nAChRs (Table 1). For wild-type nAChR, the K_app for ACh was 30 ± 8 µM. For the segment C mutant nAChRs, there was a significant rightward shift of K_app only for the Cys substitution at Y198C. The mutant nAChRs with substituted Cys adjacent to Tyr-198 at either Pro-197 or Leu-199 were characterized by leftward shifts of K_app, whereas for the other substitutions studied in this segment, the K_app values were shifted <2-fold compared with wild-type. The Cys substitutions in segment E (106-113) were also well tolerated. The largest shifts of K_app were seen for the L109C (K_app = 100 µM) and Y111C (K_app = 14 µM) mutants, whereas for substitutions at each of the other positions, K_app for ACh was within a factor of 2 of wild-type.

Modification of Substituted Cysteines within Binding Site Segment C. For oocytes expressing wild-type or mutant nAChRs, the response to ACh was measured at a concentration close to K_app. Oocytes were then exposed to MTSET (200 µM), MTSEA (1 mM), or maleimide-PEO-biotin (1 mM) (Fig. 1) for 5 s in the absence of agonist; after a wash of 1 to 2 min, the ACh response was remeasured. Representative current traces are shown for wild-type and segment C mutant nAChRs treated with MTSET (Fig. 2A), and summary data for each of the mutants are presented for the effects of MTSET, MTSEA, and maleimide-PEO-biotin (Fig. 2B). We were particularly interested in determining whether thiocholine tethered at positions other than Y198C in segment C would result in covalent activation. However, treatment with MTSET for 5 s resulted in irreversible inhibition of the ACh response by >75% for the T196C, P197C, and D200C mutant nAChRs, and a smaller inhibition of the I201C mutant that increased with longer reaction times (see later). The ~10% inhibition of the D195C or L199C mutants was less than that seen for wild-type nAChR and was not indicative of modification of the substituted Cys. Treatment with MTSEA also resulted in irreversible inhibition of ACh current responses for those mutants sensitive to MTSET. Modification with maleimide-PEO-biotin (1 mM, 5 s) significantly inhibited the ACh responses for the same mutants and, in addition, it inhibited irreversibly the D195C nAChR mutant.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Effects of MTSET, MTSEA, and Maleimide-PEO-Biotin on nAChRs containing Cys substitutions in binding site segment C. A, current responses (microamperes) of nAChRs to ACh were determined before and after a 5-s application of 200 µM MTSET. ACh test concentrations were near Kapp: (WT, 10 µM; D195C, 30 µM; T196C, 30 µM; P197C, 3 µM; Y198C, 1000 µM; L199C, 3 µM; D200C, 30 µM; I201C, 30 µM). Horizontal scale bar is 5 s. B, the mean change in current was determined by testing oocytes with at least three applications of a half-maximal concentration of ACh before and after a 5-s application of 200 µM MTSET (), 1 mM MTSEA (), or 2 mM maleimide-PEO-biotin (). Bars represent the mean change in current ± S.D. from experiments on at least three oocytes. Percentage change in current was calculated as: [(Iafter MX / Ibefore  1)] × 100.

Modification of Substituted Cysteines within Binding Site Segment E. Cys substitutions at 106 to 113 were similarly tested for their sensitivity to MTSET and MTSEA (Fig. 3). Summary data show that a 5-sec exposure to MTSET (200 µM) or MTSEA (1 mM) inhibited ACh responses for the N107C mutant by ~30 and 75%, respectively, whereas both compounds inhibited the L109C response by ~90%. Effects at the other positions tested were not sufficiently different from wild-type to make any conclusions about their accessibility. Exposure of these mutants to maleimide-PEO-biotin did not yield any additional information about residue accessibility (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of MTSET and MTSEA on nAChRs containing Cys substitutions in binding site segment E. MTSET () and MTSEA () current inhibition was determined as in Fig. 2B. ACh test concentrations, which were near Kapp, were 30 µM except for L109C, which was tested at 100 µM. Percentage current inhibition was defined as: [1  (Iafter MX / Ibefore)] × 100. Each bar represents the mean ± S.D. from experiments on at least three oocytes.

Reaction Rate Constants. The rates of reaction were measured to determine the effect of full modification at a particular residue in response to a specific reagent and to give us the kinetic information necessary to design protocols for subsequent dTC protection experiments. For the Cys mutants inhibited irreversibly by MTSET or MTSEA, the rates of reaction with mutant nAChRs were determined by measuring the response to ACh after increasing reaction times and for various reagent concentrations (Fig. 4). For oocytes expressing Y93C, Y198C, and E57C nAChRs, ACh responses were inhibited by >90% after full modification (Sullivan and Cohen, 2000). In contrast, even after complete modification, the ACh current response was not fully inhibited for a number of the segment C and E Cys mutants characterized here. For T196C and I201C mutant nAChRs, ACh current responses were maximally inhibited 30 to 50% by MTSEA, whereas P197C and D200C responses were inhibited by 75% and 95%, respectively (Fig. 4A). For substitutions in segment E, MTSEA treatment inhibited N107C responses by 80%, whereas it inhibited L109C responses by >90% (Fig. 4B), similar to the level of inhibition seen for the E57C mutant. MTSET treatment of T196C nAChRs resulted in maximal inhibition of 80% (Fig. 4C), compared with the maximal inhibition of 30% seen after reaction with MTSEA. MTSET also more fully inhibited responses at P197C than MTSEA. MTSET treatment inhibited to the same extent as MTSEA at D200C (>90%) and I201C (50%). For substitutions in the -subunit, MTSET fully inhibited the E57C and L109C nAChR, whereas for the N107C nAChR, maximal inhibition was only 50% (Fig. 4D).

View larger version (39K): [in this window] [in a new window]   Fig. 4.   Kinetics of modification of nAChRs with Cys substitutions in binding site segments C, D, or E by MTSEA, MTSET, and MBTA. Symbols represent the fraction of residual current response plotted as a function of cumulative modification time. ACh responses were measured before and after each 5-s application of reagent and were normalized to the initial response. Plots were fit with single exponential functions as described under Materials and Methods to give the first-order rate constant 1/ (s1) and the fractional response remaining after full modification, I. A, MTSEA modification rates are shown for segment C residues: T196C, 1 mM ( = 6 s, I = 0.65); P197C, 30 µM ( = 6 s, I = 0.26); D200C, 100 µM ( = 13 s, I = 0.06); I201C, 1 mM ( = 8 s, I = 0.56). B, MTSEA modification rates are shown for -subunit Cys substitutions in binding site segments D and E: E57C, 300 µM ( = 10 s, I = 0); N107C, 30 µM ( = 8 s, I = 0.13); L109C, 30 µM ( = 5 s, I = 0.09). C, MTSET modification rates are shown for segment C residues: T196C, 300 nM ( = 10 s, I = 0.18); P197C, 30 µM ( = 6 s, I = 0.08); D200C, 100 µM ( = 12 s, I = 0.06); I201C, 1 mM ( = 7 s, I = 0.37). D, MTSET modification rates for -subunit: E57C, 100 µM ( = 8 s, I = 0.07); N107C, 100 µM ( = 22 s, I = 0.18); L109C, 10 µM ( = 5 s, I = 0.09). E, MBTA modification rates are shown for segment C residues: T196C, 150 nM ( = 6 s, I = 0.36); P197C, 100 µM ( = 26 s, I = 0.03); D200C, 3 µM ( = 11 s, I = 0); I201C, 1 mM (not fit). F, MBTA modification rates for -subunit segment D and E residues: E57C, 100 µM ( = 12 s, I = 0); N107C, 1 mM (not fit); L109C, 3 µM ( = 17 s, I = 0.15). Parameter uncertainties were 5 to 20% for  and 1 to 15% for I.

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View this table: [in this window] [in a new window]   TABLE 2 Modification reaction rate constants of mutant nAChRs by MTSEA, MTSET, and MBTA Reaction rate constants, k (mM1 sec1), were determined as described under Materials and Methods and in Fig. 4 from experiments on 2 to 8 oocytes.

Effects of Alkylation on ACh Dose Response. Analysis of the kinetics of MTSET modification of the mutant nAChRs revealed that after full modification, ACh responses were inhibited by 90% at some positions (Y93C, P197C, D200C, E57C, L109C) but not at others (T196C, I201C). For the latter positions, it was clear that the tethered thiocholine modified the ACh response but did not prevent ACh binding. Additional experiments were carried out to determine whether, at the other positions, tethered thiocholine acted as an irreversible antagonist or alternately as a modifier of ACh binding and/or gating. We characterized ACh dose response curves before and after modification to determine whether the inhibition seen at a fixed concentration of ACh resulted from reduction only of the maximal response or also from a modification of K_app. If modification resulted in a nAChR no longer capable of being activated by ACh, then any ACh induced currents could result only from remaining unmodified nAChRs, and the response after exposure to MTSET would be characterized by a decreased maximal current without change of K_app. For example, modification of Y93C or E57C nAChRs by MTSET or MTSEA resulted in reductions of maximum current without change of K_app (Sullivan and Cohen, 2000). If after modification, ACh was still able to gate the ion channel, but with either the binding or gating altered, then the response could be characterized by a shift of K_app.

View larger version (39K): [in this window] [in a new window]   Fig. 5.   ACh dose response curves before (filled symbols) and after (open symbols) reaction with MTSET. A, data were fit as described under Materials and Methods. Responses were normalized to the maximal response for each oocyte. A, D195C (solid lines): Kapp = 20 µM, nH = 1.5; after 200 µM MTSET, 5 s: Kapp = 20 µM, Imax = 0.82. T196C (dotted lines): Kapp = 18 µM, nH = 2.0, after 1 mM MTSET, 15 s: Kapp = 41 µM, Imax = 0.59. B, P197C (solid lines): Kapp = 3.4 µM, nH = 1.2; after 1 mM MTSET, 10 s: Kapp = 22 µM, Imax = 0.84. L199C (dotted lines): Kapp = 3.2 µM, nH = 1.2; after 1 mM MTSET, 10 s: Kapp = 4.9 µM, Imax = 0.89. C, D200C (solid lines): Kapp = 44 µM, nH = 2.0; after 1 mM MTSET, 10 s: Kapp = 103 µM, Imax = 0.16. I201C (dotted lines): Kapp = 28 µM, nH = 1.9; after 1 mM MTSET, 10 s: Kapp = 29 µM, Imax = 0.72. D, E57C (solid lines): Kapp = 44 µM, nH = 2.0; after 100 µM MTSET, 5 s: Kapp = 47 µM, Imax = 0.34. L109C (dotted lines): Kapp = 116 µM, nH = 1.4; after 200 µM MTSET, 5 s: Kapp = 400 µM, Imax = 0.29. Each point represents the mean ± S.D. for at least three measurements. Parameter uncertainties were 5 to 20% of Kapp or nH and <10% for Imax. Values of nH were unchanged after modification, with the exception of E57C, for which nH decreased from 2.0 to 1.4.

dTC Protection Experiments. From our previous work and the experiments presented here, we identified accessible residues in ACh binding site segments A (Y93C and N94C), C (D195C, T196C, P197C, Y198C, D200C, and I201C), D (E57C), and E (N107C and L109C). However, the fact that modification of the substituted cysteines led to altered ACh responses does not establish that these positions actually contribute to the structure of the ACh binding site. The altered responses could result from an allosteric modification of the structure of the binding site or from a perturbation of the conformational transition necessary for channel gating. If MTSET is within the agonist binding site when it reacts with a substituted Cys, then that reaction should be inhibited by the presence of a reversible agonist or antagonist that is bound in proximity to that position.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Competitive antagonist protection of MTSET alkylation of reactive Cys residues. To measure the degree of protection by 10 µM dTC, current responses to ACh at a concentration near Kapp were measured before and after 10 µM dTC was coapplied for 5 s with a concentration of MTSET known to cause 50 to 80% inhibition. The same concentration of MTSET was then applied for 5 s in the absence of dTC, again using ACh test pulses to measure the extent of inhibition. The degree of protection was defined as [1  (% InhibitiondTC/MTSET / % InhibitionMTSET)] × 100. Current traces are shown from oocytes expressing wild-type and nAChRs with substitutions in segments A (Y93C), D (E57C), and E (L109C) (left) and segment C (T196C, P197C, D200C, and I201C) (right). Horizontal scale bar is 5 s.

A Homology Model of the T. californica nAChR Binding Site. The studies described above were carried out before the publication of the structure of the molluscan AChBP (Brejc et al., 2001). To facilitate discussion of our results, we developed a model of the T. californica nAChR - binding site based upon the structure of the AChBP (Fig. 7). The amino acids identified by affinity labeling and mutagenesis as contributors to the ACh binding site of the nAChR are located at each subunit interface, with amino acids of segments A, B, and C contributed from one subunit and amino acids from segments D, E, and F from the other. Secondary structure elements are identified by the ribbon representation and key binding site side chains depicted in ball and stick representation. After energy minimization of the AChBP model backbone containing the primary sequences of the extracellular regions of the T. californica nAChR subunits, no significant movement was noted for the structures containing the amino acids of binding site segments A-E. As discussed in "Materials and Methods", the size of the insertions in the segment F region of the - or -subunit prohibits any confident prediction of the position of those insertions, and we do not depict amino acids from this region in our model of the binding site. With that caveat, the most prominent structure changes between the ACh binding site of the AChBP and the model of the T. californica nAChR binding site were due to single amino acid substitutions, primarily within segment E. 

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Stereo representation of the T. californica nAChR agonist binding site at the interface between the - and -subunits. A homology model of the T. californica nAChR was constructed from the known three-dimensional structure of the molluscan AChBP. The -sheet regions of the model are denoted by the numbering system for the AChBP (1, 2, etc.). A stereo representation of the ACh binding site is presented in ball and stick representation of side chains identified by affinity labeling or mutational analyses, including the Cys substitutions described in this report. The amino acids identified are in binding site segments A (Tyr-93 and Asn-94, gold), B (Trp-149 and Tyr-151, red), C [Tyr-190, Cys-192/193 disulfide (yellow), Thr-196, Tyr-198, and Asp-200, blue], D (Trp-55 and Glu-57, green), and E (Asn-107, Leu-109, Tyr-111, Tyr-117, and Leu-119, magenta). A section of the segment F ribbon is included (gray) as well as Lys-34 on 1 (brown). An ACh molecule (dotted Connolly surface) is shown within the site. After energy minimization, the ACh nitrogen, represented by the cyan sphere, is equidistant (~5 Å) from the aromatic side chains of Trp-55, Trp-149, Tyr-190, and Tyr-198 and 6 Å from Tyr-93. The acetyl group protrudes into the opening between segments C and E with the carbonyl oxygen oriented toward Leu-119. The arrow denotes the likely route of ligand access.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have determined the accessibility for modification of individually substituted cysteines within segments C (195-201) and E (106-113) and have further assessed their accessibility in the presence of the competitive antagonist dTC. We previously identified Y93C, N94C, Y198C, and E57C as accessible for modification (Sullivan and Cohen, 2000). Similar to the results obtained for segments A and D, within segment E, only two positions (N107C, L109C) were clearly identified by our assay as accessible for modification. Within segment C, all of the positions tested demonstrated accessibility for modification, except for L199C. The pattern of protection by dTC identifies residues within the binding site likely to be in close proximity to bound dTC, and the selective protection of residues within segment C identifies the surface of a -strand projecting into the dTC/agonist binding site. With the availability of a model of the T. californica nAChR extracellular domain, based upon the AChBP structure, it becomes possible to evaluate our results to identify consistencies and differences between the model and the structure of the nAChR binding site in the absence of agonist.

Modification of Substituted Cysteines within Segments C and E. Our studies in segment C complement previous studies in which functional embryonic mouse Cys mutant nAChRs (183-197) were expressed on the cell surface and accessible for modification by a thiol-specific biotin, with the exception of Y190C (Spura et al., 2000). We found that ACh responses were readily quantified for each mutant T. californica nAChR containing Cys substitutions within 195-201, and all residues within 195-201, with the exception of L199C, were accessible for modification. For most of these positions, introduction of either a primary amine (after MTSEA reaction) or quaternary amine (after MTSET or MBTA reaction) caused an altered response to ACh rather than an irreversible inhibition of binding. For the Y93C, E57C, and Y198C receptors, earlier results indicated that covalent modification prevented the binding of ACh. Clearly, direct radioligand binding studies are required to determine the equilibrium constants for ACh binding to the modified Cys mutant nAChRs.

dTC Protection and Binding Site Structure. Because our functional assay identified modification of substituted cysteines in segments A (Y93C, N94C), C (196-201, except L199C), D (E57C), and E (N107C, L109C), we wanted to determine how dTC binding altered the accessibility of cysteines for modification. If bound dTC sterically occluded access of MTSET to a binding site Cys, the rate of reaction would be reduced in proportion to dTC occupancy. Within segment C, dTC protected substituted cysteines from alkylation at 196, 198, and 200 but it did not protect 197 or 201. This dTC protection pattern is readily explained if this portion of segment C were organized as a -strand with the side chains of 196, 198, and 200 on a common surface projecting toward the dTC/ACh binding site, as is seen in the structure of the molluscan AChBP (Brejc et al., 2001) and in the nAChR binding site model (Fig. 7, blue).