Figure 3.

Estimation of agonist k_on at WT and S193^5.42A 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 S193^5.42A with RGS4 and GIRK1/4. The rates of rise (k_obs) 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 k_on, estimated from the slopes of the linear fits to k_obs shown in (A), (B), and (C). Shown are mean k_on estimates for DA (WT: 9.70 ± 1.23 × 10⁷ s⁻¹ × M⁻¹, S193^5.42A: 3.69 ± 0.48 × 10⁴ s⁻¹ × M⁻¹), (S)-5-OH-DPAT (WT: 2.86 ± 0.31 × 10⁷ s⁻¹ × M⁻¹, S193^5.42A: 2.82 ± 0.34 × 10⁶ s⁻¹ × M⁻¹), (R)-5-OH-DPAT (WT: 8.65 ± 1.21 × 10⁵ s⁻¹ × M⁻¹, S193^5.42A: 1.95 ± 0.15 × 10⁶ s⁻¹ × M⁻¹), and p-tyramine (WT: 4.94 ± 1.15 × 10³ s⁻¹ × M⁻¹, S193^5.42A: 9.41 ± 2.34 × 10³ s⁻¹ × M⁻¹). Note the logarithmic y-axis. (E) Relations between kinetic pK_ds, derived from k_off and k_on, and pEC₅₀s, obtained from the concentration–response experiments shown in Figure 1, at WT D₂R and S193^5.42A. The correlation between pEC₅₀s and kinetic pK_ds for all four agonists at both WT and S193^5.42A mutant D₂R was statistically significant (Spearman’s r = 0.9048, p = 0.0046) for all four pairs. Data are presented as mean ± SEM.