Article Figures & Data
Figures
- Figure 1.
A view of nicotine at the α4β2 binding site. The crystal structure of α4β2 (PDB 5KXI) on the left shows the aromatic box motif, with each loop contributing to the binding site in a unique color and nicotine in gray. The schematic on the right details the hydrogen bond (red) and cation-π interaction (purple) interactions previously determined for nicotine with TrpB (α4: 149), as well as how TyrC2 (α4: 197) could interact with other agonists. TyrA (α4: 93), TyrC1(α4: 190), and TrpD (β2: 57) are shown in the crystal structure but omitted from the schematic for clarity. An alignment of each loop contributing to the box in the human nAChR family is shown at the bottom.
- Figure 2.
The structures and electrostatic potential maps of acetylcholine and nicotine are shown here for comparison to the secondary amine agonists and have been calculated with Hartree Fock 6-31G** (shown on a scale of −10, red and more negative electrostatic potential, to +150, blue and more positive electrostatic potential, kcal/mol).
- Figure 3.
Fluorination plots of all the agonists tested in this report at TrpB in α4β2. The x-axis is the predicted M06/6-31G(d,p) DFT-calculated energies between a sodium ion and each side chain (labeled) in the gas phase as described in Davis and Dougherty (2015). The y-axis is the log of the fold-shift in EC50. Each agonist tested showed a linear trend and, therefore, demonstrated a functional cation-π interaction at TrpB, as previously seen with acetylcholine and nicotine. Data plotted for varenicline are from Tavares et al. (2012).
- Figure 4.
Fluorination plots of all the agonists tested in this report at TyrC2 in α4β2. The x-axis is the predicted M06/6-31G(d,p) DFT-calculated energies between a sodium ion and each side chain (labeled) in the gas phase as described in Davis and Dougherty (2015). The y-axis is the log of the fold-shift in EC50. Each agonist tested shows a linear trend and, therefore, demonstrates a functional cation-π interaction with TyrC2, a result not previously seen with acetylcholine or nicotine.
- Figure 5.
Representative traces from dose-response experiments with a variety of agonists, noncanonical amino acid substitutions, and Imax values.
Tables
TrpB EC50 (μM) nH Imax (μA) Fold shift N Trp 0.023 ± 0.0009 1.4 ± 0.06 0.22–1.46 1 15 F1Trp 0.043 ± 0.0008 1.3 ± 0.03 0.12–1.2 1.8 11 F2Trp 0.052 ± 0.001 1.2 ± 0.04 0.05–0.62 2.2 14 F3Trp 0.13 ± 0.003 1.2 ± 0.03 0.11–1.41 5.5 12 F4Trp 0.15 ± 0.007 1.1 ± 0.05 0.15–0.77 6.6 14 TyrC2 EC50 (μM) nH Imax (μA) Fold shift N Phe 0.098 ± 0.003 1.1 ± 0.03 0.06–1.08 1 17 F1Phe 0.14 ± 0.005 1.2 ± 0.04 0.05–0.38 1.5 12 F2Phe 1.6 ± 0.07 1.3 ± 0.06 0.05–0.57 16 9 F3Phe 3.0 ± 0.25 1.3 ± 0.11 0.07–0.18 30 7 TrpB EC50 (μM) nH Imax (μA) Fold shift N Trp 0.64 ± 0.02 1.3 ± 0.0 0.08–0.89 1 13 F1Trp 0.79 ± 0.02 1.4 ± 0.0 0.12–0.80 1.2 16 F2Trp 3.6 ± 0.1 1.5 ± 0.1 0.15–0.56 5.6 14 F3Trp 13 ± 1 1.6 ± 0.1 0.07–0.30 20 12 F4Trp 16 ± 2 1.3 ± 0.1 0.03–0.11 25 12 TyrA EC50 (μM) nH Imax (μA) Fold shift N Phe 19 ± 5 1.1 ± 0.2 0.02–0.16 1 7 F3Phe 17 ± 2 1.3 ± 0.1 0.01–0.03 0.9 6 TyrC2 EC50 (μM) nH Imax (μA) Fold shift N Phe 0.41 ± 0.03 1.2 ± 0.07 0.06–2.78 1 17 F1Phe 0.86 ± 0.06 1.2 ± 0.08 0.04–0.07 2.1 11 F2Phe 11 ± 1 0.6 ± 0.1 0.03–0.13 27 8 F3Phe 21 ± 1 1.4 ± 0.2 0.04–0.24 51 12 TrpB* EC50 (μM) nH Fold shift N Trp 0.0024 ± 0.0001 1.2 ± 0.1 1 15 F1Trp 0.0057 ± 0.0002 1.2 ± 0.1 2.4 11 F2Trp 0.0057 ± 0.0021 1.2 ± 0.1 2.4 14 F3Trp 0.027 ± 0.001 1.3 ± 0.1 11 12 F4Trp 0.056 ± 0.005 1.1 ± 0.1 23 14 TyrC2 EC50 (μM) nH Imax (μA) Fold shift N Phe 0.0014 ± 0.0002 1.3 ± 0.14 0.04–0.14 1 10 F1Phe 0.0020 ± 0.00009 1.5 ± 0.09 0.02–0.08 1.4 8 F2Phe 0.011 ± 0.00097 1.2 ± 0.11 0.02–0.1 8.1 12 F3Phe 0.027 ± 0.0016 1.1 ± 0.06 0.02–0.09 19 8 TrpB EC50 (μM) nH Imax (μA) Fold shift N Trp 1.7 ± 0.1 1.3 ± 0.1 1.33–9.37 1 13 F1Trp 4.6 ± 0.2 1.3 ± 0.1 0.27–0.9 2.8 16 F2Trp 11 ± 0.7 1.3 ± 0.1 0.04–0.11 6.4 8 F3Trp 26 ± 2 1.5 ± 0.1 0.05–1.28 16 16 F4Trp 44 ± 4 1.2 ± 0.1 0.95–1.51 27 8 TyrC2 EC50 (μM) nH Imax (μA) Fold shift N Phe 3.3 ± 0.3 1.2 ± 0.1 0.02–1.42 1 15 F1Phe 5.5 ± 0.3 1.0 ± 0.1 0.04–0.11 1.7 11 F2Phe 31 ± 3 1.2 ± 0.1 0.03–0.26 9.6 10 F3Phe 35 ± 2 1.4 ± 0.1 0.02–0.14 11 12
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