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FIGURE 1. Chemical structures of α7 nAChR allosteric modulators. On the basis of experimental data obtained in the present study, compounds have been classified as allosteric agonists, desensitizing (type I) PAMs, nondesensitizing (type II) PAMs, NAMs, or SAMs. Compounds differ only in the pattern of methyl substitution at a single aromatic ring (designated Ar in the figure). Information concerning the ratio of cis-cis- and cis-trans-diastereoisomers obtained during synthesis is provided in the supplemental materials.
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FIGURE 2. Pharmacological properties of allosteric agonists on α7 nAChRs expressed in X. laevis oocytes.
A, bar graph illustrating agonist responses observed with 100 μm of each compound. Responses are normalized to the average response obtained upon application of acetylcholine alone at a maximum effective concentration (3 mm). Data are means ± S.E. of 3–15 independent experiments (Table 1). B, representative recordings are shown illustrating responses to the application of acetylcholine (3 mm) and of the allosteric agonists (100 μm). The horizontal lines indicate the duration of agonist applications. Responses have been normalized to their peak response. C, concentration-response data are presented for a range of concentrations of 2,3,4MP-TQS (open squares), 2,4MP-TQS (open circles), 4MP-TQS (open diamonds), and acetylcholine (closed circles). Data are means ± S.E. of at least three independent experiments, each from different oocytes. Data are normalized to agonist-induced responses induced by the maximal effective concentration.
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FIGURE 3. Pharmacological properties of type I PAMs on α7 nAChRs expressed in X. laevis oocytes.
A, representative recordings are shown illustrating responses to the application of acetylcholine (100 μm) alone or to a maximal concentration of the PAM (PentaMP-TQS 10 μm; 2,3,4,6MP-TQS 100 μm) preapplied for 10 s and then co-applied with acetylcholine (100 μm). Solid horizontal lines indicate the application of acetylcholine, and dotted horizontal lines indicate the application of the PAM. B, concentration-response data are presented for a range of concentrations of 2,3,4,6MP-TQS (open diamonds) and PentaMP-TQS (open circles) on responses evoked by a submaximal (EC50) concentration of acetylcholine with wild-type α7 nAChRs. The PAM was preapplied for 10 s and then co-applied with acetylcholine (100 μm). Data are means ± S.E. of at least three independent experiments, each from different oocytes. Data are normalized to a submaximal (EC50) concentration of acetylcholine (100 μm).
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FIGURE 4. Pharmacological properties of type II PAMs on α7 nAChRs expressed in X. laevis oocytes.
A, representative recordings are shown illustrating responses to the application of acetylcholine (100 μm) and a maximal concentration of the PAM (100 μm) preapplied for 10 s and then co-applied with acetylcholine (100 μm). Solid horizontal lines indicate the application of acetylcholine, and dotted horizontal lines indicate the application of the PAM. Responses have been normalized to their peak response. B, concentration-response data are presented for a range of concentrations of 2MP-TQS (open triangles), 2,3,5MP-TQS (open squares), and 3,5MP-TQS (crosses) on responses evoked by a submaximal (EC50) concentration of acetylcholine (100 μm) with wild-type α7 nAChRs. The PAM was preapplied for 10 s and then co-applied with acetylcholine (100 μm). Data are means ± S.E. of at least three independent experiments, each from different oocytes. Data are normalized to a submaximal (EC50) concentration of acetylcholine (100 μm).
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FIGURE 5. Pharmacological properties of antagonists (NAMs) on human α7 nAChRs and mouse 5HT3ARs expressed in X. laevis oocytes.
A, representative recordings are shown illustrating responses with wild-type α7. In the top panel, Orthosteric agonist indicates the application of acetylcholine (100 μm) and a maximal concentration of the antagonist (200 μm) preapplied for 10 s and then co-applied with acetylcholine (100 μm). In the bottom panel, Allosteric agonist indicates the application of 2,4MP-TQS (10 μm) and a maximal concentration of the antagonist (200 μm) preapplied for 10 s and then co-applied with 2,4MP-TQS (10 μm). Solid horizontal lines indicate the application of the agonist, and dotted horizontal lines indicate the application of the NAM. B, concentration-response data are presented illustrating the ability of 2,3,6MP-TQS (filled circles) and 2,6MP-TQS (open triangles) to inhibit responses evoked by a submaximal (EC50) concentration of acetylcholine (100 μm) on wild-type α7. The antagonist was preapplied for 10 s and then co-applied with acetylcholine (100 μm). Data are means ± S.E. of at least three independent experiments, each from different oocytes. Data are normalized to a submaximal (EC50) concentration of acetylcholine (100 μm). C, both 2,3,6MP-TQS and 2,6MP-TQS are noncompetitive antagonists of acetylcholine. Concentration-response data are presented for a range of concentrations of acetylcholine acting on wild-type α7 nAChRs in either the absence (filled circles) or presence of 20 μm 2,3,6MP-TQS (open circles) or 40 μm 2,6MP-TQS (open triangles). In all cases the antagonist was preapplied for 10 s and then co-applied with acetylcholine. Data are means ± S.E. of at least three independent experiments, each from different oocytes. Data are normalized to acetylcholine (3 mm). D, in the top panels, representative recordings are shown illustrating responses on α7M253L to the application of acetylcholine (100 μm) (left panel) and a maximal concentration of the antagonist (200 μm) preapplied for 10 s and then co-applied with acetylcholine (100 μm) (right panel). In the bottom panels, the same protocol was used for the 5-HT3A receptors except CPBG (1 μm) was used as the agonist instead of acetylcholine. Solid horizontal lines indicate the application of CPBG, and dotted horizontal lines indicate the application of the PAM.
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FIGURE 6. Pharmacological properties of SAMs on α7 nAChRs expressed in X. laevis oocytes.
A, representative recordings are shown illustrating responses to the application of acetylcholine (100 μm) (left panel) and a maximal concentration of the SAM (100 μm) preapplied for 10 s and then co-applied with acetylcholine (100 μm) (right panel). B, representative recordings are also shown illustrating responses to the application of 2,4MP-TQS (10 μm) (left panel) and a maximal concentration of the SAM (100 μm) preapplied for 10 s and then co-applied with 2,4MP-TQS (100 μm) (right panel). Solid horizontal lines indicate the application of acetylcholine, and dotted horizontal lines indicate the application of the SAM. C, concentration-response data are presented illustrating the ability of 2,4,6MP-TQS to have no effect on a submaximal (EC50) concentration of acetylcholine (100 μm) (□) but to inhibit responses evoked by a submaximal (EC50) concentration of 2,4MP-TQS (10 μm) (●). The compound was preapplied for 10 s and then co-applied with either agonist. Data are means ± S.E. of at least three independent experiments, each from different oocytes. Data are normalized to a submaximal (EC50) concentration of either acetylcholine (100 μm) or 2,4MP-TQS (10 μm). D, the α7 nAChR transmembrane mutation L247T converts SAMs (2,3,5,6MP-TQS and 2,4,6MP-TQS) into agonists. Representative recordings are shown illustrating responses to the application of acetylcholine (10 μm) (left panel), 2,3,5,6MP-TQS (10 μm) (middle panel), and 2,4,6MP-TQS (10 μm) (right panel) on α7L247T nAChRs. Solid horizontal lines indicate the application of acetylcholine, and dotted horizontal lines indicate the application of the SAM.
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FIGURE 7. Competition radioligand binding on tsA201 cells transiently co-transfected with human α7 nAChR cDNA and C. elegans RIC-3 cDNA. Equilibrium radioligand binding was performed with [3H]α-bungarotoxin (10 nm) in the presence of varying concentrations of competing ligands (MLA or allosteric modulators). No significant displacement of [3H]α-bungarotoxin was observed with any of the 19 methyl-substituted compounds up to the maximum texted (100 μm). (Note that for simplicity, data are shown only for two allosteric modulators (2,3,6MP-TQS and 2,6MP-TQS).) In contrast to the findings with allosteric modulators, complete displacement of [3H]α-bungarotoxin was observed with the orthosteric ligand MLA. Data are means of triplicate samples from a single experiment, from three independent experiments ± S.E. Data are normalized to the maximal [3H]α-bungarotoxin binding.
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