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Figure 1. Xylazine is
a competitive antagonist of α7 nAChR. (a) Representative
traces showing xylazine (XYL) inhibition of currents elicited by acetylcholine
(ACh) in Xenopus oocytes expressing
α7 nAChR. (b) XYL concentration-dependent inhibition of α7
nAChR currents elicited by ACh. Inhibitory response is expressed as
the fraction of current induced in the presence (I) of the indicated concentrations of XYL relative to that in the
absence (I0) of XYL. The data fit to the
Hill equation and resulted in XYL half-maximal inhibitory concentrations
(IC50) as shown in the figure. (c) ACh concentration-dependent
curves for α7 nAChR activation in the absence and presence of
XYL, showing a right shift of EC50 with increased concentrations
of XYL. These data were used to generate the inserted Schild plot,
for which the Gaddum/Schild nonlinear regression fitting resulted
in a Schild slope of 0.95 (R2 = 0.98),
indicating XYL is a competitive antagonist. The fitting also shows
a binding affinity of 8.4 μM for XYL onto α7 nAChR. All
data in (c) are normalized to the maximum current of α7 nAChR
activated with 3 mM ACh. All data in (b,c) are reported as the mean
± SEM from n ≥ 7 Xenopus oocytes from four donors. (d) Representative view of xylazine (red)
binding to the orthosteric site in the extracellular domain (ECD)
of α7 nAChR based on AutoDock.
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Figure 2. Representative
trace showing α7 nAChR currents activated
by acetylcholine (ACh, 50 μM) and potentiated by PAMs (turmeric,
DMC, ivermectin, and PNU120596). All PAMs were applied 30 s earlier
(preincubation) than the agonist ACh. The result was confirmed by
different Xenopus oocytes (n = 5) expressing α7 nAChRs.
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Figure 3. α7 nAChR PAMs can offset the inhibitory action of
xylazine
(XYL). (a) The α7 nAChR PAMs, including DMC (0.5 μM),
ivermectin (IVM, 1 μM), and PNU-120596 (PNU, 0.5 μM),
significantly increased the ACh potency (smaller EC50).
DMC also increased the efficacy with a higher maximum plateau of I/I3 mM ACh than that
from the control. (b) The PAMs could effectively offset the inhibitory
effects of XYL (10 μM, red). The α7 nAChR PAMs were used
with a 30 s preincubation. Data in (a,b) are normalized to the α7
nAChR current activated by 3 mM ACh, fit to the Hill equation, and
reported as the mean ± SEM from n ≥ 7 Xenopus oocytes and four different donors.
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Figure 4. Xylazine inhibition of [Ca2+]in transients
elicited by choline (Cho) activation of endogenous α7 nAChRs
in PC12 cells loaded with Calbryte 520 AM. (a) Representative traces
of [Ca2+]in fluorescent intensity stimulated
by different concentrations of Cho. The fluorescence intensity resulting
from the first 10 mM Cho application in each cell is defined as Fref for normalizing fluorescence intensities
under different Cho concentrations in (b). (b) [Ca2+]in fluorescence responses as a function of Cho concentrations
in the absence (black) and presence of xylazine (XYL, 10 μM,
pink). The data fitting to the Hill equation resulted in Cho EC50 of 2.50 ± 0.10 or 2.71 ± 0.15 mM in the absence
or presence of XYL, respectively. Data points are presented as mean
± SEM (n ≥ 130 cells). Most error bars
are smaller than symbol sizes. Extra sum-of-squares F test [F(3,2396) = 1275, p <
0.0001] suggests a significant XYL inhibitory effect on Cho-stimulated
[Ca2+]in transients.
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Figure 5. DMC potentiates [Ca2+]in signals upon activation
of α7 nAChRs by choline and counteracts the xylazine inhibitory
effect. (a) Compared to the control (black), DMC (0.4 μM) significantly
potentiated Cho-stimulated [Ca2+]in transients
(blue) with Cho EC50 = 1.83 ± 0.12 mM and Emax = 1.19 ± 0.03. The statistical significance
of DMC potentiation is based on analyses of the extra sum-of-squares F test [F(3,1925) = 136.7, p < 0.0001]. (b) DMC (0.4 μM) reversed the xylazine inhibition
(orange) and brought the [Ca2+]in transients
back to the control level, as indicated by the extra sum-of-squares F test [F(3,2339) = 0.2126, p = 0.8877]. The data were fit to the Hill equation. Data points are
presented as mean ± SEM (n ≥ 130 cells).
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Figure 6. Xylazine effects on α4β2 nAChRs. The assemblies
of
(α4)2(β2)3 and (α4)3(β2)2 nAChRs show distinctly different responses
to ACh in their ACh concentration-dependent curves. The presence of
10 μM xylazine induces a right shift of the concentration-dependent
curves and elevation of ACh EC50 values in both assemblies.
All data are normalized to the maximum current of (α4)2(β2)3 or (α4)3(β2)2 activated with 1000 μM ACh. All data are reported as the mean
± SEM from n ≥ 13 Xenopus oocytes from two donors.
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Figures S1. α-Bungarotoxin (BTX, 125 nM, an α7nAChR-selective antagonist) completely
inhibited the ACh-induced current from Xenopus oocytes expressing α7nAChR, confirming that
the observed current results from α7nAChR activation.
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Figures S2. Representative images of live PC12 cells load with Calbryte™ 520 AM under
different perfused solutions. (a) Choline (Cho) activation of α7nAChR and xylazine (XYL)
inhibition as shown in changes of cell fluorescence: (left) Basal fluorescence, (middle) after
applying 10 mM Cho, (right) after applying 10 mM Cho + 10 μM XYL. (b) DMC potentiation of
Cho-stimulated [Ca2+]in transient: (left) Basal fluorescence, (middle) after applying 10 mM Cho,
(right) after applying 10 mM Cho + 0.4 μM DMC. (c) DMC recovers XYL-induced decrease of
[Ca2+]in transient: (left) Basal fluorescence, (middle) after applying 10 mM Cho, (right) after
applying 10 mM Cho + 10 μM XYL + 0.4 μM DMC. Note that images in each row were obtained
from the same population of cells.
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Figures S3. Xylazine inhibits [Ca2+]in increases elicited by acetylcholine (ACh) activation of
endogenous α7nAChRs in PC12 cells loaded with Fura-2AM in a dose-dependent manner.
Increases in [Ca2+]in transients are plotted as a function of ACh concentrations, measured by the
F340/F380 ratio of Fura-2 AM (MilliporeSigma) loaded in PC12 cells. The presence of xylazine
(10 μM or 30 μM) significantly inhibits the ACh-elicited [Ca2+]in transients. The α7nAChRselective antagonist MLA (5μM) largely, but not completely, inhibits the [Ca2+]in transients,
reflecting that unlike choline, ACh is a non-specific agonist to α7nAChRs and ACh-elicited
[Ca2+]in transients also result from receptors other than α7nAChR. The data fitting to the Hill
equation resulted in EC50 and Emax (maximum efficacy). Data points are presented as mean ± SEM
(n ≥ 70 cells). Two-way ANOVA mixed-effects analysis shows significant xylazine effects (10 μM,
p=0.01; 30 μM, p<0.0001).
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Figures S4. Evaluation of autofluorescence resulting from DMC. DMC-produced
autofluorescence in the absence of receptor agonists is measured by the normalized fluorescence
(F/Fref) as a function of DMC concentrations, where F is the autofluorescence at a given DMC
concentration in the absence of Cho; and Fref is the fluorescence at 10 mM Cho without DMC.
Data are presented as mean ± SEM (n = 249 cells from 8 different dishes). Note that 0.4 μM DMC
was selected for our experiments involving DMC because this concentration showed minor
autofluorescence (0.23) but effective potentiation of α7nAChR while counteracting xylazine’s
inhibitory effects (Fig. 5).
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Figures S5. Xylazine docking results on α7, α4β2, and α3β4 nAChRs and extracellular
domain sequence alignment of these receptors. (a) AutoDock1 reveals that xylazine (molecule
in magenta) binds to the orthosteric ligand site in the extracellular domain (ECD) of these receptors.
The average docking energies are marked. (b) The sequence alignment of the ECDs covering the
orthosteric ligand site. The α7 residues within 3.5 Å of xylazine (marked in red) and aligned with
corresponding residues in α4, β2, α3, and β4. The non-identical residues embracing the xylazine
binding site (marked in green) likely contribute to the varied docking energies among different
subtypes of nAChRs. A lower docking energy of xylazine in α7nAChR than α4β2nAChRs is
consistent with the experimental finding that xylazine introduces a greater change of EC50 in the
agonist ACh in α7nAChR. In addition, the docking results and sequence alignment suggest that
xylazine is also likely to inhibit α3β4 nAChRs as it does on α7nAChR and α4β2nAChRs.
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Figures S6. DMC potentiates α7nAChRs but not α4β2nAChRs. DMC, preincubated for 30 s
before co-application with acetylcholine (EC10, 30 μM for α7 and 1 μM for α4β2), potentiates
currents of α7nAChRs in a dose-dependent manner but does not show potentiation effects on
(α4)2(β2)3 and (α4)3(β2)2. All data were normalized to the current elicited by acetylcholine in the
absence of DMC (I0) and presented as mean ± SEM. Each data set was generated from n ≥ 7
oocytes from two different donors.
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Figures S7. Ivermectin is not a PAM for α4β2nAChRs. (a) Representative current traces of
(α4)3(β2)2 and (α4)2(β2)3 elicited by acetylcholine (ACh, 1 μM) in the absence and presence of
ivermectin (IVM, 1 or 10 μM) or DMC (1 or 10 μM) or PNU120596 (PNU, 0.5 or 10 μM) that is
a known α7-specific PAM but not a PAM of α4β2nAChRs.
2 As expected, PNU120596 does not
produce potentiation of (α4)3(β2)2 and (α4)2(β2)3 currents. Consistent with the results shown in
Figures S6, DMC shows no potentiation of (α4)3(β2)2 and (α4)2(β2)3. Like PNU120596 and DMC,
ivermectin does not show positive modulation of (α4)3(β2)2 and (α4)2(β2)3, either. (b) The
sequence alignment of the transmembrane domain embedding the ivermectin binding site in
α7nAChRs shows differences between key α7 residues binding residues and those at equivalent
positions of α4 or β2. These differences may impede ivermectin binding to (α4)3(β2)2 and
(α4)2(β2)3. The results shown in (a) were confirmed by different Xenopus oocytes expressing
(α4)3(β2)2 (n = 5) and (α4)2(β2)3 (n = 3).
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