Chapter 2. Cation- π analysis using fluorinated Trp derivatives
2.3 Discussion
2.3.6 Behavior of tertiary and quaternary nAChR agonists
As is readily evident from Figure 2.12, nicotine deprotonation is unable to explain the line shape seen in the plot of log EC50 versus cation-π binding ability. N-methyl- nicotinium is incapable of being deprotonated, but it gives very similar results to nicotine itself. Certainly, there is no regular increase in EC50 with increasing fluorination that characterizes a direct electrostatic interaction with Trp149.
0 0.2 0.4 0.6 0.8 1
0.01 0. 1 1 10 100 0.01 0. 1 1 10 100
Normalized Current
[Nicotine] (µM)
16 20 24 28 32
0 0.5 1 1.5 2
log [EC50/EC50(wt)]
Cation-π Binding (kcal/mol) 0
0.2 0.4 0.6 0.8 1
Normalized Current
[N-Me-Nicotine] (µM)
Figure 2.12 Electrophysiological analysis of nicotine and its quaternary analog. Left panel:
Nicotine dose-response relations for βγ L9’S nAChR suppressed at α149 with Trp (O), F–Trp ( ), F2-Trp (◊), F3-Trp (), and F4-Trp (). Center panel: Dose-response relations for N-Me- nicotinium for βγ L9’S receptors suppressed with Trp (O), F–Trp ( ), F2-Trp (◊), F3-Trp (), and F4-Trp () at α149. Right panel: Plot of log [EC50/EC50(wt)] for ACh (O), nicotine ( ), and N-Me- nicotine () at the nAChR versus the calculated cation-π binding energy of the series of fluorinated Trp derivatives.
Indeed, nicotine does not experience such an interaction with any of the aromatic amino acids comprising the binding site, as inferred from the AChBP crystal structure.
Figure 2.2 shows that substitution of γ55/δ57 with Trp analogs of radically different electrostatic character gives very little difference in EC50. Additionally, earlier studies carried out on Tyr93 and Tyr198 do not show shifts characteristic of nicotine-tyrosine cation-π interactions.16 Substitution of Tyr190 with even very modestly different unnatural analogs proved impossible, however, so we are unable to rule out the possibility that nicotine interacts with this residue.
Where does this leave us as far as comparing the means of molecular recognition in the two receptors? The expectation for 5-HT3R was that modulation of the charge density of the cationic center would track with the slope of log EC50 versus cation- πbinding energy. It stands to reason that the more energetic the interaction between cation and aromatic, the more binding would depend on it. In the simplest case of gas- phase binding of ions to a series of fluorinated Trp's, the slope for Li+ would be greater than Na+, which would be greater than K+, and so on.10 Clearly, the situation in the binding site is more complicated than this.
Where alkali metal ions are approximately spherical, the charge density of serotonin analogs is highly anisotropic. In fact, shape selection is usually considered to be extremely important in the binding of small molecules in hydrophobic protein active sites.44 The 5-HT3R has evolved to bind serotonin, which is a primary amine. Even though 5-HTQ binds and gates with channel with identical potency and efficacy to 5-HT, it is safe to assume that the binding site is optimized for the smaller serotonin cation. The fluoro-Trp-based assay may, in fact, be reporting on extremely subtle differences in positioning, as the electrostatic interaction between cation and πsystem is strongly distance-dependent. In the case where the natural agonists are being compared, as in 5–HT with 5-HT3R and ACh with nAChR, it may well be the case that the comparison is significant. Here, the systems are comparable, in that the binding site has been optimized for best fit. The implication that the productive binding mode for 5-HT is more dependent on the electrostatics of Trp183 than is ACh on Trp149 may well be taken to mean that non-cationic interactions with the receptor play a greater role in nAChR.
This interpretation is in some ways consistent with what was observed for TMA by Wenge Zhong.19 This very simple cation is an agonist of nAChR, but with very low potency. Of all agonists tested, in fact, its potency is the lowest. However, it exhibits a cation-π interaction with Trp149. (Figure 2.13) In other words, productive binding of TMA involves the quaternary ammonium group nestled up against the face of Trp149.
From the perspective of the aromatic box, when TMA gets this close, it is quite similar to ACh. The acetyl component of ACh doesn't have room to fit in the box, and probably extends out of the box altogether during productive binding. If TMA looks so similar to ACh at its ammonium head group, then, is it safe to conclude that the additional potency
of ACh comes from interactions between the acetyl group and another part of the binding site? It certainly seems so. This is also in accord with what is observed for nicotine and its N-methylated derivative.
0 0.5 1 1.5 2
16 20 24 28 32
log [EC 50/EC 50(wt)]
Cation-π Binding (kcal/mol)
Figure 2.13 Plot of log [EC50/EC50 (wt)] for ACh (O), norACh ( ), and TMA () at the nAChR versus the calculated cation-π binding energy of the series of fluorinated Trp derivatives.
Like TMA, these compounds are significantly less potent than ACh. But unlike TMA, they do not appear to come quite so close to the aromatics of the box during their productive binding mode. Indeed, no cation-π interaction is detected with any part of the aromatic box. Nonetheless, the compounds are reasonably efficacious. The conclusion is that the channel can be opened by compounds relying on interactions outside of the aromatic box. The most surprising result of all, a priori, is probably the behavior of norACh. (Figure 2.13) The backbone of this compound is identical to ACh. Any non- cationic interaction available to ACh should be equally available to norACh. Indeed, the cationic center of norACh is an even better cation-π binder than the quaternary ammonium of ACh. However, norACh is an agonist of amazingly low potency. It is tempting to conclude that the aromatic box of the nicotinic receptor has evolved to accommodate a quaternary ammonium, and anything smaller is rejected. Until a tertiary compound is discovered which displays an unequivocal cation-π interaction, this argument may even be maintained.
However, it is important to note that nicotine certainly opens the channel without apparently coming in van der Waals contact with the aromatic box. And productive binding is primarily what one is interested in, as far as understanding channel function.
The neuronal receptors, for which nicotine is a far more potent agonist than the muscle receptor, will present a very interesting system for study. It is tempting to speculate that these receptors contain aromatic boxes which are more accommodating of nicotine's pyrrolidinium cation and allow it to behave like ACh in all respects. It is also the case that there are hundreds of interesting nicotinic agonists which remain unstudied by this method, including the secondary amine epibatidine. It is highly likely that high- resolution structural data of AChBP may be able to shed further light on the varieties of agonist binding modes.