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. 2019 Jan;144:172-183.
doi: 10.1016/j.neuropharm.2018.10.032. Epub 2018 Oct 23.

Amphetamine Enantiomers Inhibit Homomeric α7 Nicotinic Receptor Through a Competitive Mechanism and Within the Intoxication Levels in Humans

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Free PMC article

Amphetamine Enantiomers Inhibit Homomeric α7 Nicotinic Receptor Through a Competitive Mechanism and Within the Intoxication Levels in Humans

Daniel R Garton et al. Neuropharmacology. .
Free PMC article

Abstract

Amphetamine-type stimulants (ATS) are the second most consumed illicit drug worldwide and lack good treatments for associated substance use disorders, lagging behind other addictive drugs. For this reason, a deeper understanding of the pharmacodynamics of ATS is required. The present study seeks to determine amphetamine (AMPH) enantiomers' effects on the homomeric α7 nicotinic acetylcholine receptor (α7 nAChR). Here we have shown that AMPH enantiomers bind to the α7 nAChR and competitively inhibit acetylcholine responses. Our in silico docking analysis suggests that AMPH binds close to the β7 strand of the B-loop of a chimera comprising of the human α7 nAChR and the acetylcholine binding protein from Lymnaea stagnalis. This may inhibit the required movement of the C-loop for channel opening, due to steric hindrance, providing a structural mechanism for its antagonist effect. Finally, we have shown that, in α7 nAChR full knockout mice, the behavioral response to D-AMPH is attenuated, providing direct evidence for the role of α7 nAChRs on the physiological response to D-AMPH. Importantly, D-AMPH exerts these effects at concentrations predicted to be pharmacologically relevant for chronic methamphetamine users and during binges. In conclusion, our data present new findings that implicate the α7 nAChR on the pharmacodynamics of ATS, which may be important for behavioral responses to these drugs, indicating a potential role for α7 nAChRs in ATS substance-use disorders.

Keywords: Alpha 7 nicotinic acetylcholine receptor; Alpha 7 upregulation; Amphetamine-induced locomotion; Amphetamine-type stimulant; In silico docking analysis; Pharmacological characterization.

Conflict of interest statement

Disclosure:

The authors declare no conflict of interest.

Figures

Fig. 1:
Fig. 1:
Amphetamine enantiomers inhibit α7 nAChRs through a competitive mechanism. A. [3H]-MLA competition binding assay on hippocampal tissue. Binding experiments were performed on ice (n = 3), and the inhibition constant (Ki) calculation model was fitted to a single binding site model using a KD of 0.45 nM for MLA. D-AMPH displaces [3H]-MLA (1.5 nM) in a concentration-dependent manner, reaching levels close to [3H]-MLA nonspecific binding at the highest D-AMPH concentration. B. Concentration−response curve for hα7R activation by Ach (1–1000 µM) and AMPH enantiomers (3–1000 µM) Left: Representative traces for hα7R macroscopic currents generated by ACh and D-AMPH on Xenopus laevis oocytes. ACh or D-AMPH were perfused for 5 s. Traces alternate in color (grey and black) for visual facilitation. Right: ACh induces hα7R currents (n = 5) in a concentration-dependent manner, while each enantiomer displays no responses (n = 8). C. Concentration−response curve for hα7R inhibition by AMPH enantiomers. Top: Representative traces for hα7R macroscopic currents generated by ACh and D-AMPH. Antagonism was tested by pre-perfusing the respective enantiomers (0.1– 300 µM, 5 s), followed by ACh (1 mM) + AMPH coperfusion (5 s). Bottom: AMPH enantiomers inhibit hα7R currents with significantly different potencies. Extra sum−of−squares F test, F(1,115) = 8.4, P = 0.005. D. Current−voltage (IV) relationship for hα7R inhibition by AMPH enantiomers. Top: Representative traces for the voltage ramp applied during ACh (55 µM) and ACh + D-AMPH (30 µM) coperfusion. A nondesensitizing ACh concentration was perfused with or without the respective AMPH enantiomer for 22 s. The ramp was applied 10 s after ligand exchange onset. Bottom: AMPH enantiomers do not rectified the IV relationship at negative potentials as expected for open channel blockers (D-AMPH: n = 5–6, two-way ANOVA, treatment F(1,90) = 0.82, P = 0.37; L-AMPH: n = 6, two-way ANOVA, treatment F(1,100) = 0.22, P = 0.64). Each data point is represented as mean ± SEM.
Fig. 2:
Fig. 2:
D-Amphetamine displays surmountable inhibition on α7 nAChR endogenously expressed in primary cultures from the CA1 region of the hippocampus. A. Concentration−response curve for mα7R inhibition by AMPH enantiomers. Left: Representative traces for MLA-sensitive, mα7R macroscopic currents recorded from primary cultures of the CA1 region. ACh (2 mM) was air-pressured applied (150 ms, 16 p.s.i.; arrowhead) through a glass micropipette (1−2 µm tip) 20 µm away from the neuron. Right: AMPH enantiomers inhibit mα7R currents with no differences in potencies. Extra sum−of−squares F test, F(1,42) = 2.8, P = 0.1, n = 3–5 cells/concentration. A n = 3 was only used for L-AMPH at 730 µM, which displayed total mα7R inhibition like D-AMPH at 730 µM (n = 5) and experiments in oocytes for the hα7R. Antagonism was tested by bath applying one of the enantiomers (0–730 µM, 10 minutes), while ACh was air-pressured puffed every minute. B. ACh vs D-AMPH competition analysis. Left: Representative traces for mα7R currents. ACh, D-AMPH, and ACh + D-AMPH solutions were rapidly exchanged (~20 ms) by a fast perfusion system, and cells were perfused for 2 s. Right: Increasing ACh concentrations reduce D-AMPH inhibition until reaching a concentration with minimal inhibition (two-way ANOVA, interaction F(4,33) = 11.0, P = 0.0001; Fisher’s LSD posttest, *P < 0.05, **P < 0.01, ***P < 0.001; n = 4–5 cells/condition). D-AMPH concentrations represents the IC33, IC66, and IC90. Each data point is represented as mean ± SEM.
Fig. 3:
Fig. 3:
D-Amphetamine binding to an α7/AChBP chimera is stabilized by hydrogen bonding to Ser144’s backbone carbonyl groups. A. Side and top views for the volume of the α7/AChBP chimera binding site (cube) that was selected for docking analysis. The cube center was superimposed over Trp143 and the edge dimensions are 100 Å. The dotted box demarcates the β strands (β9 and β10) comprising the C-loop, and the + and – symbols signify the principal and complementary sides of the binding site. B. Nitrogen atoms from the D-AMPH molecules display a high degree of occurrence near Ser144’s carbonyl group. The docking protocol was run ten times using the “Apo” α7/AChBP conformation, with residues delineating the nicotine binding site allowed to rotate freely, except for Trp145. Cluster 1: 21 orientations (mint green), Cluster 2: 4 orientations (dark gray), Cluster 3: 19 orientations (banana mania), Cluster 4: 27 orientations (yellow), Cluster 5: 1 orientation (green), Cluster 6: 1 orientation (orange), Cluster 7: 38 orientations (pink salmon), Cluster 8: 5 orientations (light violet), Cluster 9: 4 orientations (tangerine). C. Docked D-AMPH molecules residing close to the β7 strand of the B-loop may prevent C-loop displacement by steric hindrance. Left: Orientations from the most populated cluster, Cluster 7. Part of the C-loop was removed to improve visualization. Center: Orientations in Cluster 7 were stabilized by coincident hydrogen bonds with Tyr91 and Ser142. The orientation with the lowest root mean square (RMS, 1.95 Å, −6.09 kcal/mol) within this cluster is displayed. RMS for each orientation was calculated using the RMSD for each combination in the clusters. Dashed lines show hydrogen bonds with Tyr 91 and Ser142 (yellow), the π-σ interaction with Trp145 (purple), and the π-π stack interaction with Tyr184 (magenta). No interaction with water was observed. Right: van der Waals radius for the orientation with the lowest RMS. This orientation interacts (cutoff distance 3.9 Å) with all residues involved in nicotine binding, except Leu116. The contacts made by Tyr91, Trp145, Tyr184, and Tyr 191 may prevent the C-loop displacement required for channel opening. Scale: partial atom charge. D. 1- methyl-2-pyridin-3-ylethylamine is an α7 nAChR agonist with negligible efficacy. Left: Nicotine, (±)-AMPH, and (±)-MPEA structures. Center: Representative traces for hα7R macroscopic currents generated by ACh and (±)-MPEA on Xenopus laevis oocytes. Traces alternate in color (grey and black) for visual facilitation. Right: (±)-MPEA hardly induces macroscopic currents on hα7R. ACh (1 mM) and (±)-MPEA (0–1000 µM) were perfused for 4 s. The mean peak current was 1278 ± 131 nA for ACh. For (±)-MPEA: 2.33 ± 0.26 nA at 0 µM, 3.09 ± 0.34 nA at 300 µM, and 10.2 ± 1.3 nA at 1000 µM. One-way ANOVA: F(5,41) = 25.4, P < 0.001; Dunnett’s posttest (control: 0 µM (±)-MPEA): *P < 0.05; n = 8 oocytes. Data points represent mean ± SEM.
Fig. 4:
Fig. 4:
D-amphetamine administration induces a reduced locomotion increase in α7 nAChR full knockout mice and does not upregulate α7 nicotinic receptors in wild type mice. A. α7 nAChR KO mice displays no changes in locomotion when exposed to an open field for the first time. Mice were acclimated to the chamber for 90 minutes (habituation phase), followed by the treatment injection and locomotion assessment for 90 minutes (treatment phase). The Box and Whisker graph covers the total data range. Habituation, WT: 12499 ±679 (n = 10), KO: 11141 ± 694 (n = 12), Student’s t-test P = 0.18. B. α7 nAChR KO mice displays reduced locomotion to an acute D-AMPH dose. Horizontal locomotion was assessed during the treatment phase with saline (second day) or D-AMPH (1.5 mg/kg, i.p., third day). motion. Saline, WT: 5714 ± 845, KO: 4947 ± 643; D-AMPH, WT: 17601 ± 3982, KO: 10271 ± 1556; two-way repeated measure ANOVA: treatment P < 0.0001, interaction P = 0.06; Fisher’s LSD posttest: *P < 0.05, **P < 0.0005. C. Increase in horizontal locomotion concomitantly occurs with a reduction in vertical movement. Saline, WT: 729 ± 147, KO: 609 ± 84; D-AMPH, WT: 261 ± 71, KO: 257 ± 75; two-way repeated measure ANOVA: treatment P < 0.0001; Fisher’s LSD posttest: *P < 0.01, **P < 0.005. D. D-AMPH does not modify α7 nAChR expression in the prefrontal cortex, striatum and hippocampus. Mice were injected twice per day with saline or D-AMPH (2.0 mg/kg, i.p.) for 10 days. α7 nAChR expression was assessed by [3H]-MLA binding assay (10 nM) on ice. Bars represent mean ± SEM.

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