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Figure 1. Structures,
potencies, recovery from antagonism, and binding kinetics
of SWR-1-8, SV-III-130, and SWR-1-14 at the D2R. A) Structures
of SWR-1-8, SV-III-130, and SWR-1-14. B) Concentration–response
relationship for DA-induced GIRK activation in oocytes coexpressing
D2R, RGS4, and GIRK1/4 channels (EC50 = 33 nM; n = 4). C) Concentration–response curves for the
inhibition of the GIRK response to 100 nM DA by SWR-1-8 (n = 3), SV-III-130 (n = 3), and SWR-1-14 (n = 3). D) Recovery of D2R-mediated GIRK activation
by DA after antagonism by SWR-1-8 (n = 8 for 1 μM
DA and n = 5 for 100 μM DA), SV-III-130 (n = 11 for 1 μM DA and n = 12 for
100 μM DA), and SWR-1-14 (n = 7 for 1 μM
DA and n = 4 for 100 μM DA). Graphs show mean
GIRK current traces normalized to the maximal response evoked by 1
μM DA (40 s), followed by 30 μM SWR-1-8 or SWR-1-14 or
1 μM SV-III-130 coapplied with 1 μM DA (125 s) and finally
reversed by 1 μM (thick dotted line) or 100 μM (thick
solid line) DA (400 s). Thin lines indicate SEM. E) Extent of recovery
from antagonism following application of 1 or 100 μM DA (data
from experiments shown in panel D). F) Rate of recovery from antagonism
following application of 1 or 100 μM DA (data from experiments
shown in panel D). G) Observed rates of GIRK response decay, kobs, upon application of varying concentrations
of antagonist in the presence of 100 nM DA during 125 s; n = 3–7. Data shown are means ± SEM *; p < 0.05, ***; p < 0.001, and ****; p < 0.0001, Student’s t test.
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Figure 2. Curve-shift GIRK activation assay of D2R antagonism
upon coapplication of SV-III-130 and DA. A) Assay principles; 1 μM
DA elicits a full agonist response (left, red arrow). The subsequent
response amplitude in the presence of variable concentrations of SV-III-130
and DA, following 500 s coapplication (right, green arrow), was normalized
to the control response elicited by 1 μM DA. In the example
shown, 1 μM SV-III-130 coapplied with 100 μM DA; n = 3 oocytes. Thick lines represent mean normalized currents,
whereas thin lines indicate SEM. B) Current amplitude at the end of
the 500 s coapplication period, normalized to the instantaneous maximum
response amplitude in the same oocyte and plotted against DA concentration,
for varying concentrations of SV-III-130 or control. n = 3–7 oocytes per data point. Data shown are means ±
SEM.
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Figure 3. Potencies, recovery from antagonism, and binding
kinetics of SV-III-130
at the V91A, L94A, and E95A mutant D2R. A) DA potency at
V91A (EC50 = 21 nM, n = 3–4), L94A
(EC50 = 41 nM, n = 3–7), E95A (EC50 = 15 nM, n = 3–4), W100A (EC50 = 323 nM, n = 3), and WT (EC50 = 33 nM, n = 4) D2R. B) SV-III-130 potency
at the V91A (n = 4), L94A (n = 3),
E95A (n = 7), and W100A (n = 3)
D2R. C) Recovery of activation by DA at the V91A (n = 3 for 1 μM DA and n = 4 for 100
μM DA), L94A (n = 13 for 1 μM DA and n = 13 for 100 μM DA), E95A (n =
5 for 1 μM DA and n = 3 for 100 μM DA),
and W100A (n = 4 for 10 μM DA and n = 4 for 300 μM DA) mutant D2R following antagonism
by SV-III-130. GIRK current traces normalized to the maximal response
evoked by 1 μM (10 μM for W100A) DA. Thick lines represent
mean normalized currents, whereas thin lines indicate SEM. D) Extent
of recovery upon application of 1 or 100 μM (10 and 300 μM
for W100A) DA following antagonism by 30 μM (V91A and E95A),
3 μM (W100A), and 1 μM SV-III-130 (L94A; data from experiments
shown in panel C). E) Rate of recovery following application of 1
or 100 μM DA (10 and 300 μM for W100A; data from experiments
shown in panel C). F) Observed association rates, kobs, which allowed for calculation of association rates
for SV-III-130 at the V91A, L94A, and E95A D2R. n = 3–7. G) Clustering of kinetic Kd relative to Ki for SWR-1-8,
SWR-1-14, and SV-III-130 at the WT receptor and SV-III-130 at the
V91A, L94A, and E95A mutant receptors, as indicated. Data shown are
means ± SEM *; p < 0.05, **; p < 0.01, and ***; p < 0.001, Student’s t test.
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Figure 4. Simulations of three-
and four-state ligand binding at a receptor.
A) Scheme depicting a surmountable antagonist ligand (L) binding to
the receptor (R), in competition with the agonist (A; DA). B) Scheme
depicting induced-fit insurmountable antagonist ligand binding, where
RL* represents an irreversibly bound antagonist ligand. C, D, E) Simulation
of response recovery from antagonism by SWR-1-8 (C), SV-III-130 (D),
and SWR-1-14 (E) at WT D2R, with 1 or 100 μM DA during
the recovery phase. The response is assumed to be proportional to
the fraction of the agonist-bound state; RA. F) Simulated recovery
of WT D2R activation by DA after prolonged, 400-s antagonism
with SV-III-130. G) Experimental recovery of WT D2R activation
by DA after prolonged, 400-s antagonism with SV-III-130. H) Simulated
curve-shift assay with SV-III-130 at WT D2R, plotting the
RA fraction after 500 s simulation time for different concentrations
of DA and SV-III-130, as indicated. I) Simulated concentration–response
curves for SV-III-130 antagonism at WT and L94A mutant D2R. “Normalized response†corresponds to the RA fraction
after 100 s of simulation time, in the presence of 100 nM DA and varying
concentrations of SV-III-130, as indicated. The L94A mutant was simulated
by removing the fourth state, RL*, from the model. J) Simulation of
response recovery from antagonism by SV-III-130 at the L94A mutant
D2R. As in I), the three-state model is employed, using
kinetic data for SV-III-130 from the L94A mutant. Simulations of SWR-1-8
(C; kon(RL) = 340 M–1 s–1, koff(RL) = 0.017
s–1), SV-III-130 (D; kon(RL) = 860 M–1 s–1, koff(RL) = 0.007 s–1, kon(RL*) = 0.01 s–1), and SWR-1-14 (E; kon(RL) = 300 M–1 s–1, koff(RL) = 0.021 s–1) at WT D2R were conducted in the presence of 1 or 100
μM DA as agonist. kon(A) = 5 ×
106 M–1 s–1 and koff(A) = 0.17 s–1 for all
simulations at WT D2R. Parameters for the three-state model
of SV-III-130 interaction at D2R L94A; kon(RL) = 890 M–1 s–1, koff(RL) = 0.007/s, kon(A) = 5 × 106 M–1 s–1, and koff(A) = 0.21/s. koff(A) = was adjusted as necessary to yield
a Kd corresponding to the DA EC50 at D2R L94A.
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Figure 5. Structural impact of
L94A mutation on SV-III-130. A) The most populated
SV-III-130 binding mode in the D2R WT (blue) and the L94A
(orange) receptors. Upon L94A mutation, the ligand translates toward
transmembrane segment 2 as indicated by the yellow arrows. Despite
translation in the L94A mutant, SV-III-130 is primarily sandwiched
by W100 and I184 similar to the WT complex. B) Comparison of the position
of W100 between the WT and L94A receptors in terms of distance between
Cα atoms of W100 and L/A94, as well as the position of the W100
side chain center of mass in the z-dimension (perpendicular
to the membrane). Both values are significantly different between
the WT and the L94A receptors. C) Representative ligand binding mode
in the WT receptor with W100 adopting a low z value
(putative RL complex). D) Representative ligand binding mode in the
L94A mutant receptor with W100 adopting a low z value
(putative RL complex). E, F) Ligand binding modes in the WT receptor
in which W100 adopts higher z values and stacks on
top of the ligand (potentially leading to RL* complexes). In C–F),
the position of the center of mass of the W100 side chain (red circle)
in the z-dimension (perpendicular to the membrane)
is highlighted. **; p < 0.01, Mann–Whitney
U test.
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Figure 6. Curve-shift
beta-arrestin2 recruitment assay of D2R
antagonism resulting from preapplication of SWR-1-8, SV-III-130, and
SWR-1-14. Curve-shift experiments for DA-induced beta-arrestin2 recruitment
to D2R following 5 min preincubation with A) SWR-1-8, B)
SV-III-130, and C) SWR-1-14. The normalized data represent mean values
from three independent experiments (n = 3) performed
in triplicate. The curves represent the best fit to the data using
nonlinear regression analysis.
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