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Figure 1. Top views depicting α4β2 nAChR based on the X-ray structure of human (α4)2(β2)3 nAChR (PDB: 5KXI) and the chemical structures of LY2087101 and dFBr.
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Figure 2. LY2087101 potentiates low- and high-sensitivity α4β2 nAChRs. Xenopus oocytes expressing human (α4)3(β2)2 (▲) or (α4)2(β2)3 (●) nAChRs were voltage-clamped at −50 mV, and currents elicited by 10-second applications of 10 µM ACh were recorded in the absence or presence of increasing concentration of LY2087101. (A and B) representative two-electrode voltage clamp traces showing the effect of increasing concentrations of LY2087101 on ACh-induced current responses of Xenopus oocytes expressing (α4)3(β2)2 and (α4)2(β2)3 nAChRs, respectively. (C) For each recording run, peak currents response were normalized to the peak current response elicited by 10 µM ACh alone, replicas from individual oocytes were averaged. Data (Average ± SE) from 26 (▲) and 7 (●) oocytes were plotted and fit to three parameters equation (Equation 1). LY2087101 potentiation of (α4)3(β2)2 and (α4)2(β2)3 nAChRs was characterized by EC50s of 1.4 ± 0.03 and 1.9 ± 0.04 µM, and Imax of 837 ± 7 and 459 ± 34%, respectively.
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Figure 3. Effect of LY2087101 on the ACh dose-response curves of low- and high-sensitivity α4β2 nAChRs. ACh current responses of Xenopus oocytes expressing human (α4)3(β2)2 (A, △, ▲) or (α4)2(β2)3 (B, ●, ○) nAChRs were recorded in the absence of any other drug (△,○) and in the presence of 1 µM LY2087101 (▲, ●). For each ACh concentration (−/+LY2087101), peak current response was normalized to the peak current response elicited by 1 mM ACh in the same recording run. Replicas from individual oocytes were averaged and Averages ± SE for N oocytes (A, 18 (△), 8 (▲); B, 12 (○), 13 (●)) were plotted and fit to Equation 1. Values of ACh EC50/h/Imax were: for (α4)3(β2)2 nAChR (A), ACh (control), 113 ± 26 μM/0.66 ± 0.06/117 ± 6%; ACh(+1 LY2087101), 71 ± 25 μM/0.7 ± 0.1/251 ± 19%; for (α4)2(β2)3 nAChR (B), ACh (control), 1.7 ± 0.1 μM/1.2 ± 0.03/100 ± 1%; ACh (+1 LY2087101), 2.1 ± 0.1 μM/1.0 ± 0.04/189 ± 2%.
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Figure 4. LY2087101 potentiation of α4β2 nAChR requires α4 but not β2 subunits. Representative two-electrode voltage clamp traces showing the effect of LY2087101 or dFBr at 1 μM on ACh-induced current responses of Xenopus oocytes expressing α4β2, α4β4, α3β2, or α3β4 nAChR.
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Figure 5. Effect of Amino acid substitutions within α4 extracellular and transmembrane domains on LY2087101 and dFBr potentiation of (α4)3(β2)2 nAChR. Representative two-electrode voltage clamp traces showing the effect of 1 μM LY2087101 or 1 μM dFBr on ACh-induced current responses of Xenopus oocytes expressing (α4)3(β2)2 nAChRs containing an amino acid substitution at the α4 subunit extracellular domain (A) or within the α4 subunit transmembrane domain (B and C).
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Figure 6. LY2087101 concentration-dependent potentiation of WT and mutant α4β2 nAChRs. Peak ACh-induced current responses of Xenopus oocytes expressing WT or mutant (α4)3(β2)2 nAChRs in the presence of increasing concentrations of LY2087101 were normalized to the peak current elicited by 10 µM ACh alone. Shown are data (Average ± SE) from several oocytes. Data were fit to a single site model using Equation 1 and parameters (potentiation EC50 and Imax) are shown in Table 2.
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Figure 7. Effect of LY2087101 on the ACh dose-response curve of mutant α4β2 nAChRs. Currents elicited by Xenopus oocytes expressing (α4)3(β2)2 nAChRs containing point mutation within the α4 subunit in response to 10-second applications of increasing concentrations of ACh (alone (○) or +1 µM LY2087101 (●)) were recorded and normalized to peak currents elicited by 1 mM ACh alone. Replicas from the same oocyte were averaged and the Average ± SE of data from at least 3 oocytes were plotted and fit to a single site model using Equation 1. For (α4C254S)3(β2)2; (α4L256F)3(β2)2; (α4S258M)3(β2)2; (α4L260M)3(β2)2; (α4T261D)3(β2)2; (α4F316L)3(β2)2; (α4F606Y)3(β2)2; (α4G613L)3(β2)2 nAChRs, ACh Imax (Ave ± SEM) calculated from the fit of ACh concentration-response curves in the presence of 1 µM LY2087101 were 185 ± 04; 104 ± 09; 232 ± 10; 264 ± 80; 180 ± 23; 122 ± 13; 144 ± 013; 112 ± 9%, and the probability (P) that Imax in the presence of 1 µM LY2087101 were different from normalized current response to 1 mM ACh alone (Imax = 100) were <0.001, 0.878, <0.001, <0.001, 0.003, 0.475, 0.062, and 0.442, respectively.
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Figure 8. dFBr concentration-dependent potentiation of WT and mutant α4β2 nAChRs. Peak current of Xenopus oocytes expressing WT or mutant (α4)3(β2)2 nAChRs responses to 10 s application of 10 μM ACh in the absence or presence of increasing concentrations of dFBr were normalized to the peak current elicited by 10 µM ACh alone. Shown are data (Average ± SE) from at least 4 different oocytes. Data were fit to a single site model using Equation 1. Values of dFBr potentiation EC50/Imax/h were: for WT (α4)3(β2)2, 1.0 ± 0.3 μM/580 ± 42%/1.1 ± 0.2; for (α4C254S)3(β2)2, 1.5 ± 0.5 μM/430 ± 31%/0.8 ± 0.1; and for (α4L291V)3(β2)2, 2.9 ± 1.9 μM/600 ± 136%/0.8 ± 0.2. For (α4L256F)3(β2)2, (α4E308V)3(β2)2, and (α4G613L)3(β2)2, inhibition at 10 μM dFBr were ~25, 45, and 65%, respectively.
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Figure 9. Effect of dFBr on the ACh dose-response curve of mutant α4β2 nAChRs. Currents elicited by Xenopus oocytes expressing mutant (α4)3(β2)2 nAChRs in response to 10-second applications of increasing concentrations of ACh (alone (○) or +1 µM dFBr (▲)) were recorded and normalized to peak currents elicited by 1 mM ACh alone. Replicas from the same oocyte were averaged and the Average ± SE of at least 3 oocytes were plotted and fit to a single site model using Equation 1. ACh Imax in the presence of 1 µM dFBr calculated from curve fitting for (α4C254S)3 (β2)2, (α4L256F)3 (β2)2, (α4L260M)3 (β2)2, (α4E308V)3 (β2)2, (α4F316L)3 (β2)2, (α4F606Y)3 (β2)2, (α4G613L)3(β2)2 were 190 ± 08, 142 ± 08, 250 ± 36, 110 ± 06, 148 ± 12, 159 ± 07, and 157 ± 19%, and the probability (P) that Imax in the presence of 1 µM dFBr were different from normalized current response to 1 mM ACh alone (Imax = 100) were <0.001, 0.057, <0.001, 0.602, 0.031, 0.001, 0.012, respectively.
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Figure 10. LY2087101 binding sites in the transmembrane domain of (α4)3(β2)2 nAChR. Top (A) and Side (B–E) views showing LY2087101 docked into the (α4)3(β2)2 nAChR at three transmembrane pockets; two within the α4 subunit transmembrane helix bundle (Sites 1 and 2) and one at the α4:α4 transmembrane interface (Site 3) as described under computational docking analyses method section. LY2087101 is shown in yellow space-filling model (A,B) or in ball and stick format colored by elements (C–E; Carbone, gray; oxygen, red;). The nAChRs subunits are shown in ribbon with the α4 subunit that provides the (+)face of the α4:α4 interface colored in red and designated as α4 subunit A. The α4 subunit that provides the (−)face of the α4:α4 interface (colored in cyan and designated as α4 subunit B) does not exist in the crystal structure of (α4)2(β2)3 nAChR (PDB: 5KXI)6 and therefore was derived from this crystal structure by homology modeling as described previously29. The other nAChR subunits are colored in grey (B) or not shown (A) for clarity. Details of LY2087101 interactions with amino acids residues within Binding Site 1–3 are shown in C–E, respectively, with hydrogen bond interactions are shown as green dashed lines, non-bond hydrophobic interactions are shown as violet dashed lines, and unfavorable interactions as red dashed lines.
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Figure 11. dFBr binding sites in the transmembrane domain of (α4)3(β2)2 nAChR. Side views showing dFBr docked within the α4 subunit transmembrane helix bundle (Binding Site 1, A) and at the α4:α4 transmembrane interface (Binding Site 3, B). The α4 subunits are shown as ribbon with the subunit that provides the (+)face and (−)face of the α4:α4 interface are colored in red and cyan, respectively. dFBr is shown in ball and stick format colored by element whereas key amino acids side chains are shown in line format. Hydrogen bond interactions and non-bond hydrophobic interactions between dFBr and amino acids residues within Binding Site 1 and 3 are shown as green and violet dashed lines, respectively.
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