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Binding sites for agonists and competitive antagonists (nondepolarizing neuromuscular blocking agents) are located at the α–δ and α–ε subunit interfaces of adult nicotinic acetylcholine receptors. Most information about the amino acids that participate in antagonist binding comes from binding studies with (+)-tubocurarine and metocurine. These bind selectively to the α–ε interface but are differentially sensitive to mutations. To test the generality of this observation, the authors measured current inhibition by five competitive antagonists on wild-type and mutant acetylcholine receptors.
HEK293 cells were transfected with wild-type or mutant (αY198F, εD59A, εD59N, εD173A, εD173N, δD180K) mouse muscle acetylcholine receptor complementary DNA. Outside-out patches were excised and perfused with acetylcho-line in the absence and presence of antagonist. Concentration–response curves were constructed to determine antagonist IC50. An antagonist-removal protocol was used to determine dissociation and association rates.
Effects of mutations were antagonist specific. αY198F decreased the IC50 of (+)-tubocurarine 10-fold, increased the IC50 of vecuronium 5-fold, and had smaller effects on other antagonists. (+)-Tubocurarine was the most sensitive antagonist to εD173 mutations. εD59 mutations had large effects on metocurine and cisatracurium. δD180K decreased inhibition by pancuronium, vecuronium, and cisatracurium. Inhibition by these antagonists was increased for receptors containing two δ subunits but no ε subunit. Differences in IC50 arose from differences in both dissociation and association rates.
Competitive antagonists exhibited different patterns of sensitivity to mutations. Except for pancuronium, the antagonists were sensitive to mutations at the α–ε interface. Pancuronium, vecuronium, and cisatracurium were selective for the α–δ interface. This suggests the possibility of synergistic inhibition by pairs of antagonists.
THE muscle-type nicotinic acetylcholine receptor channel (nAChR) is the prototypical member of the Cys-loop ligand-gated ion channel superfamily.1 These proteins are composed of α1, β1, δ, and γ or ε subunits arranged as transmembrane pentamers: (α1)(β1)δ(α1)γ in the embryonic nAChR subtype and (α1)(β1)δ(α1)ε in the adult nAChR subtype (clockwise, as viewed from the synapse). There are several reasons why so much is known about this protein. Biochemical and structural studies take advantage of the abundance of nAChR in tissue from Torpedo electric organ.2 Electrophysiologic measurements are aided by its stability in patch clamp experiments. And the discovery3 and structure determination4 of a water-soluble analog to the extracellular domain of the receptor, acetylcholine binding protein (AChBP), has led to inferences about structure–function relations of the nAChR.5
Mutagenesis, functional, labeling, and structural studies have provided detailed information about the ligand binding site in the nAChR (fig. 1A). Binding sites for agonists and competitive antagonists are located at the α–δ and α–ε (or α–γ) subunit interfaces.** Studies have identified seven noncontinuous “loops” of amino acids that participate in agonist and competitive antagonist binding to the nAChR: loops A–C are on the α subunit (primary component of the binding site), and loops D–G are on the non-α subunit (complementary component of the binding site). The three dimensional arrangement of these loops became apparent by making analogies to the structure of AChBP.4,6 AChBP is composed of 10 β strands arranged in an immunoglobulin-like topology. Several of the loops are located between the β strands, and the loops from the primary and complementary components come together at the interface between subunits. The binding site is centered around a conserved tryptophan in loop B that provides stability for the quaternary nitrogen moiety of agonists and antagonists via a strong π–cation interaction.7 Four additional aromatic amino acids are distributed around the primary and complementary components. The C loop may act as a cap that closes upon binding of agonist and greatly decreases the agonist dissociation rate.8 The amino acids on the complementary component are not conserved between the γ, δ, and ε subunits; this leads to subunit specificity for ligand binding.
Agonists such as acetylcholine have a higher affinity for the α–δ interface compared with the α–ε interface; the extent of the difference depends on both species (Torpedo receptors exhibit a 100-fold preference for acetylcholine binding to the α–δ interface9) and subtype (fetal mouse receptors exhibit a 32-fold preference,10 and adult mouse receptors exhibit very little preference11). Both sites must be occupied for efficient opening of the channel.11 Some competitive antagonists are known to bind differently to the two sites. Metocurine, for example, has a 70- to 170-fold higher affinity for the α–ε interface compared with the α–δ interface.12,13 Occupation of just one of the sites by a competitive antagonist is sufficient to prevent channel opening. Most of the information about the residues that participate in antagonist binding comes from studies of either (+)-tubocurarine or metocurine.14 Even for these similar ligands (metocurine has three additional methyl groups; fig. 1), there are differences in sensitivities to mutations. Using computational chemistry to dock ligands to a homology model of the nAChR, Wang et al. 15 showed that (+)-tubocurarine and metocurine bind with different orientations within the α–ε interface and have different contacts with the amino acid residues there.
Although the clinical action of competitive nAChR antagonists, muscle paralysis, is a straightforward consequence of their molecular action, there are outstanding questions about the effects of nondepolarizing muscle relaxants. Specific issues include the mechanisms of tetanic fade16 and of muscle relaxant synergy.17 Although these phenomena are often ascribed to actions on presynaptic nAChRs, postsynaptic explanations may still be viable. As a first step to address these questions, we use a functional determination of current inhibition to examine competitive antagonism by (+)-tubocurarine, metocurine, pancuronium, vecuronium, and cisatracurium (fig. 1B) on mouse adult wild-type and five mutant nAChRs. We chose these mutations because they have been shown to affect metocurine and/or (+)-tubocurarine binding.13,15,18,19 The αY198F mutation is in loop C, and the εD59 mutations and εD173 mutations are in loops E and G, respectively. We used the δD180K mutation to test the strength of binding to the α–δ interface. We postulated that the antagonists would show different sensitivities to mutations but that all of them would have a higher affinity for the α–ε interface compared with the α–δ interface.
HEK293 cells (American Type Culture Collection, Manassas, VA) were transfected using either a calcium phosphate precipitate20 or a lipid based reagent (FuGENE 6; Roche Diagnostics, Basel, Switzerland). Some experiments were performed using the BOSC23 cell line, a subclone of HEK293 that exhibits higher expression levels. Identical results were obtained using both cell lines. Cells were transfected with complementary DNA (cDNA) coding for subunits of mouse muscle nAChR: α, wild type or Y198F; β, wild type; ε, wild type, D59A, D59N, D173A, or D173N; δ, wild type and cotransfected with cDNA for the α subunit of human CD8 (gift of Brian Seed, Ph.D., Professor of Genetics and Health Sciences and Technology, Harvard Medical School, Cambridge, Massachusetts), a T-cell antigen used as a marker.21,22 The AChR cDNA was cloned into the pRBG4 expression vector; the CD8 cDNA was cloned into the πH3-CD8 expression plasmid. The wild type and most of the mutant cDNAs13 were gifts of Steven Sine, Ph.D. (Professor of Physiology, Mayo Clinic, Rochester, Minnesota). DNA sequencing confirmed the constructs. Measurements were performed on cells between 1 and 4 days after transfection. For the experiments with α2βδ2 receptors, we doubled the amount of δ subunit cDNA and omitted the ε subunit cDNA in the transfection mixture.
Acetylcholine chloride (purity > 99%), (+)-tubocurarine chloride (purity 98%), and pancuronium dibromide (purity > 99%) were obtained from Sigma-Aldrich Corp. (St. Louis, MO). Metocurine iodide was synthesized from (+)-tubocurarine23 at the Chemical Synthesis Center, Department of Chemistry, Stony Brook University (Stony Brook, NY). Purity of 99% was determined by 1H-nuclear magnetic resonance. Vecuronium bromide was obtained as the clinical formulation from Bedford Laboratories (Bedford, OH), 1 mg/ml (1.8 mm) in a solution containing 2.1 mg/ml anhydrous citric acid, 1.6 mg/ml sodium phosphate, and 9.7 mg/ml mannitol. Dilutions were prepared in distilled water. The highest concentration of vecuronium used, 1 μm, contained 95 μm mannitol. Cisatracurium besylate was obtained as the clinical formulation from GlaxoSmithKline (Philadelphia, PA), 2 mg/ml (2.1 mm) in a 35% benzene sulfonic acid solution. Dilutions were prepared in distilled water. The highest concentration of cisatracurium used, 1 μm, contained 0.017% benzene sulfonic acid.
Cells were prepared for patch clamp recording by replacing the culture medium with an extracellular solution consisting of 150 mm NaCl, 5.6 mm KCl, 1.8 mm CaCl2, 1.0 mm MgCl2, and 10 mm HEPES, pH 7.3. Subsequently, 3−5 μl of polystyrene beads coated with a monoclonal antibody specific for the CD8 antigen (Dyna-beads; Dynal, Lake Success, NY) were added to the culture dish. Good expression of nAChR channels in excised patches was found for most cells having two or three beads attached.
Patch pipettes, filled with a solution consisting of 140 mm KCl, 5 mm EGTA, 5 mm MgCl2, and 10 mm HEPES, pH 7.3, had resistances of 3−6 MΩ. An outside-out patch24 with a seal resistance of 5 GΩ or greater was excised from a cell and moved into position at the outflow of a HSSE-2 rapid perfusion device (ALA Scientific Instruments, Westbury, NY). The perfusion system consisted of solution reservoirs, manual switching valves, a solenoid-driven pinch valve, and two tubes inserted into the culture dish and had a time resolution of less than 100 μs.25 One tube contained extracellular solution without agonist (normal solution); the other contained extracellular solution with 300 μm acetylcholine (test solution). In the control protocol, the patch, initially perfused with normal solution, was exposed to a series of ten 0.25-s exposures to the test solution at 5-s intervals. Manual valves were used to connect to reservoirs containing a defined concentration of competitive antagonist with or without acetylcholine. An equilibrium (+/+) protocol was performed by exposing the patch to acetylcholine plus antagonist for 0.25 s, with a 5-s interval of antagonist alone. After switching back to antagonist-free solutions, the control protocol was repeated.26 Results were accepted if the current amplitudes during the second control were 80% or more of those in the first; usually, the ratio was 95% or more. A recovery (+/−) protocol was used to determine the time needed for currents to recover after removal of antagonist. This protocol was performed with the same perfusion system by equilibrating the patch with antagonist alone for 5 s and exposing the patch to acetylcholine alone for 0.60 s as described previously.27 Currents flowing during exposure of the patch to acetylcholine were measured with a patch clamp amplifier (EPC-9; HEKA Elektronik, Lambrecht, Germany), sampled at 100 μs per point and stored on a computer. Data analysis was performed off-line as described previously.26,27 Experiments were performed at room temperature (20°− 23°C) and at a patch potential of −50 mV unless otherwise indicated.
The ratio of the peak inward current after the rapid onset phase in the presence of antagonist (Iant, +/+ protocol) to that in the average of the two control currents, I0, was calculated. This provided the fraction of receptors that were inhibited by an antagonist in the absence of acetylcholine. Concentration–response curves were generated from data obtained from experiments performed with six or seven different antagonist concentrations, each used on three to six different patches. Means and SDs are shown in the figures. Individual data points were fitted to the Hill equation:
where [ant] is antagonist concentration, IC50 is the concentration producing 50% inhibition, and nH is the Hill coefficient. For δD180K receptors, control currents were usually less than 20 pA in absolute value. In these experiments, a single concentration point (at twice wild-type IC50) was determined. The rate of recovery from antagonist inhibition (+/− protocol) was determined from 7−19 measurements of the recovery time constant for 3−5 different patches. The average and SD of the reciprocal of the (concentration-independent) time constant were calculated. This rate was interpreted to be the antagonist dissociation rate, .27 The association rate, , was calculated from .
IC50 values for mutant versus wild-type receptors were compared using an F test for nonlinear regression (Prism 4; GraphPad Software, Inc., San Diego, CA). Dissociation and association rates for mutant versus wild-type receptors were compared using a Student t test. P values less than 0.01 were considered significant because five mutants were compared with wild type for each drug. With the δD180K mutation, current levels were compared with wild type using an unpaired t test; P values less than 0.05 were considered significant.
Examples of currents in wild-type and αY198F nAChRs in the absence and presence of the five competitive antagonists are shown in figures 2A–E. Rapid perfusion of 300 μm acetylcholine elicited macroscopic currents that reached a peak within 0.3 ms and decayed due to desensitization with a time constant of 30−100 ms.22 The antagonist concentrations illustrated here were chosen to produce approximately 50% inhibition of the instantaneous inward current in wild-type receptors (left panel). In the presence of 25 nm (+)-tubocurarine (fig. 2A), 10 nm pancuronium (fig. 2C), and 20 nm vecuronium (fig. 2D), the time course of the currents was similar to that of the control. In the presence of 100 nm metocurine (fig. 2B) and 50 nm cisatracurium (fig. 2E), the current reached a plateau or exhibited a secondary increase before desensitizing. This is characteristic of competitive antagonists that dissociate from the receptor on a time scale similar to or faster than desensitization.27 For these currents, the degree of inhibition was calculated from the peak inward current reached within 1 ms of agonist application. The center panel of figure 2 shows currents from receptors containing the αY198F mutation. The antagonist concentrations in this panel are the same as those shown for wild-type receptors in the left panel. The antagonist-dependent effects of αY198F are readily apparent. The mutation caused an increase in the potency of (+)-tubocurarine (fig. 2A), a decrease in the potency of pancuronium and vecuronium (figs. 2C and D), and small changes in the potency of metocurine and cisatracurium (figs. 2B and E). The graphs in the right panel of figure 2 show how the concentration–response curves were differentially affected by the αY198F mutation.
Table 1 lists the results of fitting the concentration–response curves to the Hill equation for all combinations of antagonist and receptor. The Hill coefficients ranged from 0.8 to 1.3 with the average value being 1.01 ± 0.12. Although each antagonist has two binding sites on the receptor, functional assays, such as current inhibition, are not very sensitive to the presence of a second, low-affinity site, and the Hill coefficients are expected to be close to 1.27 The antagonist–receptor combinations producing relatively high Hill coefficients, e.g., cisatracurium–wild type, may have a low-affinity site that is only approximately 10-fold less potent than the high-affinity site.
The effects of mutations on the IC50 of the competitive antagonists are illustrated in figure 3, where ratio of the mutant IC50 to the wild-type IC50 is plotted for each antagonist. The antagonist dependence of the ratio is clear, not only for the αY198F mutation (shown in fig. 2), but also for the εD59 and εD173 mutations. Therefore, mutations at εD59 produced a 13- to 16-fold increase in the IC50 of metocurine, a 4- to 5-fold increase for cisatracurium, a 2- to 3-fold increase for (+)-tubocurarine, a 1.4-fold increase for vecuronium (only εD59A shows a significant change), and no significant change for pancuronium. The sensitivity of antagonists to mutations at εD173 showed a distinct pattern from the other mutations.
It was previously found that the α–ε interface represents the high-affinity binding site for (+)-tubocurarine and metocurine. There are few known determinants of antagonist binding on the δ subunit. Experiments with α2βδ2 receptors expressed in oocytes showed that mutations at δD180 decrease the affinity of (+)-tubocurarine with δD180K showing a 16-fold decrease28 (these receptors have two “low-affinity” sites; the binding constant derived from the inhibition of bungarotoxin binding changed from 337 nm to 5,533 nm due to the mutation). We found that transfection of BOSC-23 cells with α2βδ2D180K did not result in currents in outside-out patches. Low but finite expression was obtained when δD180K was expressed with wild-type α, β, and ε. Figure 4 compares the effect of this mutation for inhibition by competitive antagonists when each drug was applied at twice the wild-type IC50 (which produces a relative current of 0.33 in wild-type receptors). As expected, the mutation did not affect inhibition by (+)-tubocurarine or metocurine because binding to the α–ε interface dominates. However, 30 nm pancuronium, 40 nm vecuronium, and 110 nm cisatracurium decreased currents in the mutant δD180K receptors less (0.62, 0.73, and 0.59, respectively) than they did in wild-type receptors. This corresponds to a 3- to 5-fold decrease in affinity.
We also measured inhibition with (wild-type) α2βδ2 receptors that are expressed when the cDNA for the ε subunit is omitted. If the high-affinity site of an antagonist were the α–δ interface, that antagonist would produce more inhibition than for wild-type receptors. Conversely, if the high-affinity site of an antagonist were the α–ε interface, that antagonist would produce less inhibition than for wild-type receptors. The results (fig. 4) show that pancuronium, vecuronium, and cisatracurium all caused a greater inhibition of α2βδ2 receptors compared with α2βεδ receptors. Although (+)-tubocurarine and metocurine produced less inhibition of α2βδ2 receptors compared with α2βεδ receptors, the effect was not as large as would be predicted from their high selectivity for the α–ε interface.
The recovery (+/−) protocol was used to determine the dissociation rate of the antagonist from the receptor. Two examples with vecuronium are shown in figure 5. The concentration of antagonist used was 3 to 5 times the IC50 value for that receptor. The control currents are shown in light gray, and the (+/−) currents are shown in dark gray. If desensitization were not present, the time course of the (+/−) current would reflect the time it takes for vecuronium to dissociate from the receptor (the rapid perfusion system removes vecuronium from the aqueous phase within 0.5 ms). The rate of desensitization in the control current was used to calculate the fraction of channels that are either open or desensitized in the (+/−) current. The results of this numerical correction procedure are shown as black traces in figure 5. In both cases, the corrected current had a steady state value that was within 10% of the peak current in the control. The corrected current reached the steady state 3.3 times faster with 280 nm vecuronium on αY198F receptors compared with 100 nm vecuronium on wild-type receptors. For each antagonist–receptor combination, the time constant for recovery was independent of antagonist concentration (not shown).
The results of these experiments are tabulated in table 1 as the dissociation rate constant. The association rate constant was calculated from the dissociation rate and the measured IC50 value. The measured dissociation rates extended over a 260-fold range from 1.4 to 370/s. The association rates were less variable; they extended over a 12-fold range from 0.7 to 8.3 × 108/m/s. Figure 6 shows that mutations affected the dissociation and association rates in different ways. For example, mutations at εD173 increased the IC50 (decreased the potency) of (+)-tubocurarine by increasing the dissociation rate. In contrast, mutations at εD59 increased the IC50 of (+)-tubocurarine primarily by decreasing the association rate. The 2-fold increase in the IC50 of pancuronium by αY198F was the result of a 4.4-fold increase in dissociation combined with a 2.2-fold increase in association. With cisatracurium, nearly all of the mutations affected both the association and dissociation rates. Interestingly, the αY198F mutation caused a large decrease in both association and dissociation, such that there was only a small change in the IC50 of cisatracurium.
These results extend our knowledge of where competitive antagonists bind on the nAChR in several ways. First, we examined a range of antagonists with different structures. Second, we used a functional assay to insure that binding is concomitant with current inhibition. Third, we considered mutations in both the α–ε and α–δ interfaces. Finally, we examined not only equilibrium inhibition but also the kinetics of inhibition.
We characterized the inhibition produced by competitive antagonists (table 1). Our results for the IC50 of antagonists to wild-type adult mouse nAChR are in good agreement with published values. Fletcher and Stein-bach12 studied channels in a stably transfected fibroblast cell line. Electrophysiologic measurements resulted in IC50 values of 11, 54, 129, and 139 nm for pancuronium, (+)-tubocurarine, metocurine, and atracurium, respectively (they did not study cisatracurium). Measurements on transfected frog oocytes by Paul et al.29 produced IC50 values of 5.5, 9.9, and 43.4 nm for pancuronium, vecuronium, and (+)-tubocurarine, respectively. It should be noted that both of these groups studied receptors in the whole cell activated with nonsaturating concentrations of acetylcholine (0.4 μM12 or 10 μM29) using relatively slow perfusion systems. The inherent assumption with this approach is that if the antagonists dissociate from the receptor, rebinding by the antagonist is more probable than binding by agonist (and subsequent activation of the channel). In our system, rapid (< 1 ms) application of a saturating concentration of acetylcholine (300 μm) allows measurement of the uninhibited current (and, from this, determination of the degree of inhibition) before the antagonists begin to dissociate. Actual measurement of dissociation rates (≤ 400/s; table 1) validates our approach.
The results of the mutation studies (table 1 and figs. 3 and and4)4) show that antagonists are affected differentially by mutations. The αY198F mutation in embryonic mouse nAChR was shown to have a greater effect on inhibition by (+)-tubocurarine than by pancuronium.18 Differences between (+)-tubocurarine and metocurine binding to AChBP30 and human nAChR15 were identified by Sine's group. They found that although these two antagonists differ by only three methyl groups (fig. 1), they are differentially sensitive to mutations. Our results on mouse nAChR are similar. (+)-Tubocurarine is affected by mutations at αY198, but metocurine is not. Mutations at εD59 affect metocurine more than (+)-tubocurarine, and the opposite is true for εD173. In docking simulations, Sine's group made the surprising finding that these two antagonists assume docking orientations that are rotated by 180° (AChBP30) or 60° (human nAChR15). Our results with pancuronium and vecuronium underscore the sensitive interactions between ligand and receptor. Although both antagonists are affected by the αY198F mutation, mutations in the ε subunit have small but significant effects on vecuronium and no effect on pancuronium. Inhibition by both of these antagonists is affected by the δ subunit mutation, whereas inhibition by (+)-tubocurarine or metocurine is not. Cisatracurium, which is a benzylisoquinolinium compound like (+)-tubocurarine and metocurine, is affected by mutations on the α and ε subunits similarly to metocurine. However, unlike metocurine, cisatracurium is sensitive to the δ subunit mutation and has distinct kinetic responses to mutations.
Binding constants for some antagonists have been obtained through measurements of the reduction of iodinated bungarotoxin binding. These experiments can reveal the affinity of both binding sites for the antagonists, but do not always make an association between the binding constant and a particular site. Although the slope of the electrophysiologically determined concentration–response curve is not a very sensitive way to characterize the lower-affinity site, the relatively high Hill slope for cisatracurium on wild-type receptors (table 1) can be explained if there are two binding sites with affinities of 62 ± 4 and 480 ± 180 nm, respectively.27 Table 2 summarizes what is known about the selectivity of adult mouse nAChR for competitive antagonists.
We must be cautious about making inferences about selectivity solely from mutation experiments because the lack of effect of a mutation could mean that the mutated residue is either not involved or only weakly involved in binding. Conversely, the presence of an effect by a mutation could mean that the mutated residue is involved in binding or that the mutation caused an allosteric change to affect the affinity of the antagonist. Our mutagenesis results are consistent with previous results showing that the α–ε interface as the high-affinity site for metocurine and (+)-tubocurarine. For pancuronium, vecuronium, and cisatracurium, our finding that receptors with a mutation in the δ subunit are inhibited more than wild-type receptors suggests that that the α–δ interface is the higher-affinity site. The small and absent effects of ε subunit mutations on vecuronium and pancuronium respectively also support this idea. Experiments in which the ε subunit is replaced with a second copy of the δ subunit (fig. 4) were consistent with the idea that pancuronium, vecuronium, and cisatracurium now had two high-affinity binding sites. We could not obtain precise estimates of the interface selectivity of pancuronium or vecuronium. Because cisatracurium is potently affected by mutations in the ε subunit, this antagonist is not as selective (3- to 8-fold).
Although these α2βδ2 receptors were not inhibited as strongly as wild-type receptors by (+)-tubocurarine and metocurine, this effect was not in quantitative agreement with the results of toxin binding experiments that show high selectivity of these antagonists for the α–ε interface. We do not have a simple explanation for this finding. We note, however, that α2βδ2 receptors may perturb the structure of the receptor significantly more than a single site mutation.
Our conclusion differs from the results of a recent study of adult mouse nAChR exposed to pairs of competitive antagonists.17 The results of that study suggested that (+)-tubocurarine and pancuronium compete for the same binding site. However, those experiments were performed under conditions of relatively low receptor occupancy where synergistic effects are expected to be small. We are currently performing experiments under conditions of high receptor occupancy to clarify this issue.
Our kinetic measurements provide additional information about the binding of competitive antagonists to the nAChR. The αY198F mutation caused a relatively small, 20% decrease in the IC50 of cisatracurium. However, this was the result of large changes in the kinetics of inhibition: a 3.6-fold decrease in the dissociation rate and a 2.8-fold decrease in the association rate. An equilibrium assay alone might have led to the conclusion that this amino acid plays a minor role in the interaction of cisatracurium with the receptor. One possible interpretation is that cisatracurium encounters αY198 on its journey into and out of its binding site but does not bind too closely to this residue. A similar effect was seen with the effect of this mutation on the kinetics of acetylcholine binding; both the association and dissociation rates decreased by a factor of 2, such that the overall affinity was unchanged.31 Most of the antagonist/mutation combinations we examined, however, suggest that the dissociation rate is the primary determinant of the IC50 value. There are some notable exceptions, and we are currently conducting experiments with other analogs of (+)-tubocurarine to better understand these observations.
The results of this study show that our concept of ligand binding sites on receptors must be broad. The interaction of competitive antagonists with the nAChR is strong, leading to nm binding affinities. However, even structurally similar competitive antagonists occupy different positions at the interface between receptor subunits, and some of them prefer different interfaces. Moreover, we have observed pharmacologic differences between mouse and human adult AChRs.32 This has implications for drug design. Although the amino groups are essential for binding to the acetylcholine binding site, addition of methyl groups to a parent compound may do more than to increase hydrophobicity; it may affect the orientation of the ligand within the site.30 The presence of two dissimilar binding sites means that drug design can follow two independent pathways and that pairs of drugs with opposite site preferences may act synergistically. This understanding was made possible by advances in structural and molecular biology. Increased resolution of the conformations of other receptor proteins will probably reveal similar intricacies in the binding of ligands to those proteins.
Supported by grant No. NS 045095 (to J.P.D.) from the National Institutes of Health, Bethesda, Maryland, and the Department of Anesthesiology, Stony Brook University, Stony Brook, New York. Presented in part at the Annual Meeting of the American Society of Anesthesiologists, San Francisco, California, October 14, 2003.
**Because this article concerns the muscle form of the nAChR, from this point on, the α1 subunit is referred to as α, and the β1 subunit is referred to as β.