C O MMU N I C A T I O N S
closest possible protonatable analogue of ACh, shows a pK shift
a
ao mf i∼n e1 s .u nit, noticeable, although not as large as for the tethered
These differences are interpreted as follows. We assume that
only the cationic forms of agonists can activate the receptor. For
the amine tethers and norACh, the protonatable amine can
equilibrate with the medium when at the agonist binding site and
the local microenvironment of the protein produces a pK
a
shift.
For nicotine, however, no pK shift is seen because the protonated
a
Figure 2. Tethered agonists at R149: tethered agonist efficacy as a function
of solution pH. ACh efficacy ) 1 at all pHs.
amine does not equilibrate with the medium once it has bound to
the receptor. The degree of receptor activation is then dependent
only on the amount of protonated nicotine available to bind to the
receptor, and the pH dependence of activation mirrors nicotine’s
a
normal pK .
This is the second recent line of evidence from our labs that
indicates a difference between the binding modes of ACh and
nicotine. We have recently shown that the potent cation-π
3
interaction observed between ACh and RTrp149 is not evident
for nicotine.13 Taken together, these data strongly suggest that
pharmacophore models for the muscle-type nAChR should be
expanded to include two distinct agonist binding modes: an ACh-
like mode and a nicotine-like mode.
Figure 3. Tertiary agonists: norACh and nicotine efficacy as a function
of solution pH. ACh efficacy ) 1 at all pHs.
We define the efficacy of a tethered agonist as the ratio of the
constitutive current that can be blocked by TMB-8 to the maximum
current induced by saturating concentrations of ACh (corrected for
basal conductance changes with pH). By considering only constitu-
tive current that can be blocked, we eliminate any background (non-
nAChR specific) leak current. The efficacies of TyrO3Q, TyrO3T,
and TyrO3S at R149 are shown in Figure 2. TyrO3Q’s efficacy, as
expected, is insensitive to pH changes, when corrected for basal
pH dependence. As described above, TyrO3T and TyrO3S exhibit
strong titration behavior when incorporated at R149.
Acknowledgment. We thank Dr. Lintong Li for her help. This
work was supported by the National Institutes of Health (NS-34407
and NS-11756).
Supporting Information Available: Procedures for the syntheses
1
13
of the tethered agonists and their H NMR, C NMR, and MS data;
protocols for unnatural amino acid mutagenesis and oocyte electro-
physiology have been published and are summarized; examples of
primary electrophysiological data (PDF). This material is available free
of charge via the Internet at http://pubs.acs.org.
These adjusted measurements of receptor activation provide a
straightforward way to assay the degree of protonation of a tethered
amine at the binding site. Thus Figure 2 gives a phenomenological
References
(
1) (a) Corringer, P. J.; Le Novere, N.; Changeux, J. P. Annu. ReV. Pharmacol.
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102-114.
pK
membrane integrity, we are unable to study the presumed plateau
at low pH values. Nevertheless, it is clear that the side-chain pK
a
for the side chain. Since pH e 5 compromises the oocyte
8
(2) Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; van Der
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a
of TyrO3T is e6 when incorporated at R149 of the nAChR,
(
4) Li, L.; Zhong, W.; Zacharias, N.; Gibbs, C.; Lester, H. A.; Dougherty, D.
substantially shifted from its value in free solution (∼9.3, measured
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9
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for N,N-dimethylaminopropanol). pK
a
shifts of this magnitude are
precedented. For example, Lys 115 in Clostridium acetobutylicum
acetoacetate decarboxylase has an effective pK of 6.0, shifted by
.5 units.10 Our results suggest that the agonist binding site of the
a
(6) Tethered amines were also incorporated at positions γ55/δ57 and R93.
Constitutive activity was seen at these sites, but as in previous work the
effect was smaller than at R149. See Supporting Information.
4
nAChR is relatively hydrophobic, consistent with the fact that the
binding site is primarily formed by aromatic residues.
(
7) Pappone, P. A.; Barchfeld, G. L. J. Gen. Physiol. 1990, 96, 1-22.
8) Cao, Y.; Mager, S.; Lester, H. A. J. Neurosci. 1997, 17, 2257-2266.
(
Nicotine (pK
a
) 7.8)12 is, of course, a noted tertiary amine agonist
(9) Perrin, D. D. Dissociation Constants of Orgainc Bases in Aqueous
Solution: Supplement 1972; Butterworth & Co.: London, U.K., 1972.
10) Highbarger, L. A.; Gerlt, J. A.; Kenyon, G. L. Biochemistry 1996, 35,
1-46.
(11) Cho, A. K.; Jenden, D. J.; Lamb, S. I. J. Med. Chem. 1972, 15, 391-
94.
of the nAChR, and we have measured the pH-dependent nicotine
efficacy in the present context (Figure 3). Efficacy is computed
for nontethered agonists by normalizing the current from applica-
tions of saturating agonist concentrations to the maximal ACh-
(
4
3
(
12) Seeman, J. I.; Whidby, J. F. J. Org. Chem. 1976, 41, 3824-3826.
(13) Beene, D. L.; Brandt, G. S.; Zhong, W. S.; Zacharias, N. M.; Lester, H.
induced current. Interestingly, there is no pK
the phenomenological pK is not measurably different from the
solution pK of the drug. In contrast, norACh (pK
) 8.3),11 the
a
shift for nicotine;
A.; Dougherty, D. A. Biochemistry 2002, 41, 10262-10269.
a
a
a
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J. AM. CHEM. SOC.
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