Table 1.
Kinetic parameters of [3H]dopamine uptake and [3H]CFT competition binding
Vmax and Km values from [3H]dopamine uptake experiments were obtained by fitting data to Michaelis-Menten kinetics. CFT Ki values were calculated from pIC50 values, determined by nonlinear regression analysis of [3H]CFT/CFT competition binding curves. Mean Ki values with S.E. intervals were calculated from the mean pKi ± S.E. I312F, T356M, and D421N show significant impairments in uptake kinetic parameters. T356M and D421N also have dramatically lower CFT binding affinity and significant increases in Bmax. A559V, E602G, and R615C display uptake and binding properties similar to DAT WT. Data are mean ± S.E., n = 3, and all experiments were performed in triplicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001, one-way ANOVA with Dunnett's multiple comparisons test.
| Km dopamine, mean ± S.E. | Vmax dopamine, mean ± S.E. (% WT) | KiCFT, mean (nm) (S.E. interval) | Bmax CFT mean ± S.E. (% of WT) | |
|---|---|---|---|---|
| μm | ||||
| WT | 0.84 ± 0.1 | 100 ± 14 | 7.80 (6.3, 9.3) | 100 ± 15 |
| I312F | 2.0 ± 0.2* | 50.5 ± 6.0* | 5.53 (5.16, 5.90) | 86.0 ± 7.1 |
| T356M | 2.2 ± 0.6* | 41.85 ± 5.8** | 298 (230, 390)*** | 197 ± 11*** |
| D421N | >10 × WT | Not fitted | 338 (267, 426)*** | 154 ± 16*** |
| A559V | 0.80 ± 0.09 | 104 ± 11 | 8.54 (7.6, 9.6) | 106 ± 11 |
| E602G | 0.93 ± 0.05 | 99.3 ± 13 | 6.92 (6.2, 7.7) | 82.1 ± 3.7 |
| R615C | 0.75 ± 0.02 | 102 ± 16 | 7.29 (6.2, 8.5) | 89.7 ± 11 |