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 (). 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
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 μ
or 10 μ
) 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; ) validates our approach.
The results of the mutation studies ( and and ) show that antagonists are affected differentially by mutations. The α
Y198F 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 (), they are differentially sensitive to mutations. Our results on mouse nAChR are similar. (+)-Tubocurarine is affected by mutations at α
Y198, but metocurine is not. Mutations at ε
D59 affect metocurine more than (+)-tubocurarine, and the opposite is true for ε
D173. 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 α
Y198F 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
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 () can be explained if there are two binding sites with affinities of 62 ± 4 and 480 ± 180 nm
summarizes what is known about the selectivity of adult mouse nAChR for competitive antagonists.
Interface Selectivity for the Binding of Competitive Antagonists to Adult Nicotinic Acetylcholine Receptor
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 () 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 α
Y198F 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 α
Y198 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.