|
Figure 1. Concentration-response relationships of agonists at WT D2R and S1935.42A. Concentration–response curves for GIRK activation elicited by application of DA, (S)-5-OH-DPAT, (R)-5-OH-DPAT, and p-tyramine (structures shown in panel (A)) in oocytes co-expressing GIRK1/4 subunits and RGS4 with (B) WT D2R and (C) D2R S1935.42A. (D) Agonist pEC50s at WT and S1935.42A D2R: pEC50 for DA (WT: pEC50 = 7.70 ± 0.07, n = 5; S1935.42A: pEC50 = 5.03 ± 0.04, n = 8), (S)-5-OH-DPAT (WT: pEC50 = 8.28 ± 0.08, n = 11; S1935.42A: pEC50 = 6.56 ± 0.05, n = 12), (R)-5-OH-DPAT (WT: pEC50 = 6.85 ± 0.16, n = 11; S1935.42A: pEC50 = 7.07 ± 0.08, n = 14), and p-tyramine (WT: pEC50 = 4.00 ± 0.21, n = 6; S1935.42A: pEC50 = 4.32 ± 0.12, n = 6–12). Comparison of pEC50s using two-way ANOVA yielded significant main effects of agonist (F(3, 65) = 281.8) and of the S1935.42A mutation (F(1, 65) = 139.2), as well as a significant interaction between these two factors (F(3, 65) = 79.98, p < 0.001 for each main effect). Sidak’s multiple comparisons test further revealed that the pEC50s of DA and (S)-5-OH-DPAT, but not p-tyramine and (R)-5-OH-DPAT, differed significantly between WT and mutant D2R, as indicated by asterisks; ***, p < 0.001. (E) Relative efficacies at WT D2R and S1935.42A for DA (WT: 1.00 ± 0.02; S1935.42A: 1.04 ± 0.04), (S)-5-OH-DPAT (WT: 0.52 ± 0.01; S1935.42A: 0.64 ± 0.01), (R)-5-OH-DPAT (WT: 0.11 ± 0.01; S1935.42A: 0.63 ± 0.02), and p-tyramine (WT: 0.09 ± 0.01; S1935.42A: 0.37 ± 0.01). WT and S1935.42A responses were normalized to the response evoked by 1 µM and 300 µM DA, respectively. The efficacy values were obtained from the fitted parameter Top, from the corresponding concentration-response curves (see Materials and Methods). The number of oocytes used for each condition is indicated on the bars in (D,E) and corresponds to the number recorded to generate the data points plotted in (B,C). Experiments were performed using a buffer perfusion rate of 1 mL/min. Data are presented as mean ± SEM.
|
|
Figure 2. Kinetics of GIRK current deactivation following agonist washout was used to estimate agonist koff. (A) Response decay time courses following washout of DA, (S)-5-OH-DPAT, (R)-5-OH-DPAT, and p-tyramine from oocytes co-expressing WT D2R with GIRK1/4 and RGS4. (B) Terminating the agonist-induced response by application of 1 µM haloperidol revealed similar rates of decay as observed in the agonist washout experiments presented in (A). (C) Response decay time constants following washout of DA, (S)-5-OH-DPAT, (R)-5-OH-DPAT, and p-tyramine from oocytes co-expressing S1935.42A D2R with GIRK1/4 and RGS4. (D) Estimated dissociation rate constants at WT D2R and S1935.42A for DA (WT: 0.197 ± 0.012 s−1, n = 6; S1935.42A: 0.207 ± 0.012 s−1, n = 8), (S)-5-OH-DPAT (WT: 0.028 ± 0.010 s−1, n = 4; S1935.42A: 0.096 ± 0.009 s−1, n = 6), (R)-5-OH-DPAT (WT: 0.030 ± 0.008 s−1, n = 5; S1935.42A: 0.069 ± 0.008 s−1, n = 6), and p-tyramine (WT: 0.123 ± 0.008 s−1, n = 7; S1935.42A: 0.112 ± 0.008 s−1, n = 5), determined by fitting exponential functions to the agonist washout phases. For DA and p-tyramine, the first 42 s, and for (S)- and (R)-5-OH-DPATs, the first 104 s following agonist washout were used to fit the exponential functions at WT D2R. 10 nM DA, 10 nM (S)-5-OH-DPAT, 300 nM (R)-5-OH-DPAT, and 10 µM p-tyramine were used for these experiments at the WT receptor. At D2R S1935.42A, 10 µM DA, 1 µM (S)-5-OH-DPAT, 300 nM (R)-5-OH-DPAT, and 1 mM p-tyramine were used, and the first 24 s of the washout were used to fit the exponential functions for all agonists. Experiments were performed using a perfusion rate of 4.5 mL/min. Data are presented as mean ± SEM.
|
|
Figure 3. Estimation of agonist kon at WT and S1935.42A mutant D2R. Individual concentrations of (A) DA, (B) (S)-5-OH-DPAT and (R)-5-OH-DPAT, and (C) p-tyramine were added to oocytes co-expressing WT D2R or D2R S1935.42A with RGS4 and GIRK1/4. The rates of rise (kobs) of the resulting current responses have been plotted against the corresponding agonist concentrations and linear fits (solid lines) and their 95% confidence bands (dotted lines) are shown. Experiments were performed using a perfusion rate of 4.5 mL/min. (D) Summary statistics for kon, estimated from the slopes of the linear fits to kobs shown in (A), (B), and (C). Shown are mean kon estimates for DA (WT: 9.70 ± 1.23 × 107 s−1 × M−1, S1935.42A: 3.69 ± 0.48 × 104 s−1 × M−1), (S)-5-OH-DPAT (WT: 2.86 ± 0.31 × 107 s−1 × M−1, S1935.42A: 2.82 ± 0.34 × 106 s−1 × M−1), (R)-5-OH-DPAT (WT: 8.65 ± 1.21 × 105 s−1 × M−1, S1935.42A: 1.95 ± 0.15 × 106 s−1 × M−1), and p-tyramine (WT: 4.94 ± 1.15 × 103 s−1 × M−1, S1935.42A: 9.41 ± 2.34 × 103 s−1 × M−1). Note the logarithmic y-axis. (E) Relations between kinetic pKds, derived from koff and kon, and pEC50s, obtained from the concentration–response experiments shown in Figure 1, at WT D2R and S1935.42A. The correlation between pEC50s and kinetic pKds for all four agonists at both WT and S1935.42A mutant D2R was statistically significant (Spearman’s r = 0.9048, p = 0.0046) for all four pairs. Data are presented as mean ± SEM.
|
|
Figure 4. Binding modes of studied compounds within the WT D2R and the S1935.42A mutant receptor. Simulations of (A) DA, (B) (S)-5-OH-DPAT, and (C) (R)-5-OH-DPAT in complex with the WT D2R and (D) (R)-5-OH-DPAT in complex with the S1935.42A D2R were clustered based on the root-mean-square deviation (RMSD) of the ligand. For each of the systems, the poses of the ligand within the most populated cluster are depicted in licorice. For each of the binding modes, the studied position 1935.42, as well as the residues that formed polar interactions with the ligand, are also shown in licorice. Polar interactions are highlighted by red lines.
|
|
Figure 5. (R)-5-OH-DPAT forms a meta-stable binding mode with S1935.42. (A) Clustering simulations of (R)-5-OH-DPAT in complex with WT D2R, based on the RMSD of the ligand, reveal two binding modes. The main binding mode was maintained over 56% of the simulation frames (green) and a meta-stable binding mode was maintained over 15% of the simulation frames (red). (B,C). For each of the binding modes, the studied position S1935.42, as well as residues that form polar interactions with the ligand, are shown. These polar interactions are highlighted with red lines. (D) A model explaining the slow Koff values observed for the unbinding of (R)-5-OH-DPAT from WT D2R. Before dissociating from the receptor (purple conformation), (R)-5-OH-DPAT bound in the main binding mode (green) assumes a meta-stable binding mode (red). When bound in this meta-stable binding mode, (R)-5-OH-DPAT can revert to the main binding mode or proceed to an unbound conformation. Hence, the dissociation of (R)-5-OH-DPAT is effectively slowed down by S1935.42. In comparison, at the S1935.42A mutant receptor, the absence of the meta-stable binding mode permits fast exchange between the bound and unbound conformations of (R)-5-OH-DPAT.
|
|
Supplementary Figure S1
|
|
Supplementary Figure S2
|
|
Supplementary Figure S3
|
|
Supplementary Figure S4
|
|
Supplementary Figure S5
|
